Compositions for and methods of identifying antigens

ABSTRACT

Replicable libraries having discrete members in defined locations for screening for antigens to a pathogenic organism are provided. Also provided are methods for using such libraries as well as a specific antigen, CT788, which induces T-cell activation during a  Chlamydia  infection.

STATEMENT AS TO FEDERALLY FUNDED RESEARCH

The United States Government has a paid-up license in this invention andthe right in limited circumstances to require the patent owner tolicense others on reasonable terms as provided for by the terms ofgrants AI039558 and AI055900 awarded by the National Institutes ofHealth.

BACKGROUND OF THE INVENTION

Excitement around vaccine technologies has renewed over the past fewyears, driven by the emergence of new disease threats to humanity, thereemergence of previously curable diseases in the Western World such asTB, the threat of bioterrorism, and increasing evidence that cancers canbe treated by vaccination, as long as the right antigens can be found.Infectious diseases still remain as one of the leading causes ofmorbidity and mortality worldwide, killing more than 13 million youngadults and children annually. TB alone is responsible for 2 milliondeaths annually, while it is estimated that combined over 3 millionindividuals die from malaria and AIDS each year. New emerging orreemerging infectious diseases, such as SARS or Avian flu, also pose acontinual threat to global world health. Many infectious diseases, suchas malaria, TB, AIDS, SARS, and influenza are caused by intracellularpathogens that are capable of growing and spreading directly withinhuman cells. Because intracellular pathogens grow sequestered withinhost cells, the humoral (antibody) immune response is often ineffectivein generating protective immunity.

When they work, vaccines are one of the most effective ways ofpreventing and treating disease. Unfortunately, many research andclinical vaccine programs have a low probability of success because,prior to the present invention, there was no way to screen all possibleantigens or predict which ones will be effective.

Thus, there is a need for new strategies to develop effective vaccinesfor the treatment of infectious diseases and cancer.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a replicable libraryincluding at least 20 (e.g., 30, 40, 50, 60, 70, 80, 90, 100, 125, 150,200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 2000, 2500,3000, 4000, or 5000) discrete members in defined locations, where (a)the members of the library each include a cell or virus including afirst polynucleotide encoding at least a portion of a polypeptideencoded by the genome of a pathogenic organism other than the cell orthe virus, the first polynucleotide operably linked to a promoter, and(b) the library includes polynucleotides encoding a portion of at least10% (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%) ofthe polypeptides encoded by the genome (i.e., of the proteome) of thepathogenic organism.

In a related second aspect, the invention provides a replicable libraryincluding at least 10 (e.g., 15, 20, 30, 40, 50, 60, 70, 80, 90, 100,125, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500,2000, 2500, 3000, 4000, or 5000) discrete members in defined locations,where (a) the members of the library each include a cell or virusincluding a first polynucleotide encoding at least a portion of apolypeptide encoded by the genome of a pathogenic organism other thanthe cell or virus, the first polynucleotide operably linked to apromoter, (b) the members each include fewer than 24 (e.g., 23, 22, 21,20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2)different polynucleotides each encoding at least a portion of apolypeptide encoded by the genome of a pathogenic organism, and (c) thelibrary includes polynucleotides encoding at least 10% (e.g., 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%) of at least portions ofthe polypeptides encoded by the genome (i.e., of the proteome) of thepathogenic organism.

In either of the first two aspects of the invention, the portion of thepolypeptide may have at least 50%, 60%, 70%, 80%, 90%, 93%, 95%, 98%, or99% sequence identity to the corresponding portion of the polypeptideencoded by the genome of the pathogenic organism. Further, each memberof the library may include a single polynucleotide encoded by the genomeof the pathogenic organism. Finally, the pathogenic organism may be abacterium, a virus, or a fungus.

In a third aspect, the invention provides a replicable library includingat least 10 (e.g., 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150,200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 2000, 2500,3000, 4000, or 5000) discrete members, where the members each include afirst cell or virus including a polynucleotide encoding a polypeptide,or a portion or a fragment thereof, differentially expressed within aneoplastic cell as compared to the corresponding normal cell, thepolynucleotide operably linked to a promoter, and the library includespolynucleotides encoding at least portions of 5% (e.g., 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%) of the polypeptidesdifferentially expressed within the neoplastic cell as compared to thecorresponding normal cell. The portion of the polypeptide may have atleast 50%, 60%, 70%, 80%, 90%, 93%, 95%, 98%, or 99% sequence identityto the corresponding portion of the polypeptide expressed in neoplasticcell. Each member of the library may contain fewer than 50, 40, 30, 20,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 polynucleotides eachencoding a portion of a different polypeptide differentially expressedin the neoplastic cell.

In any of the above three aspects, the virus may be a phage. The cell orfirst cell may be a bacterium (e.g., E. coli). The bacterium or virusmay further include a second polynucleotide encoding a polypeptide, suchas a pore-forming protein (e.g., LLO), not naturally expressed in thebacterium. Alternatively, the first polynucleotide may further encode asecond polypeptide such as a pore-forming protein (e.g., LLO). Each ofthe first polynucleotides may further include a first tag sequence,where each of the polynucleotides encode a fusion protein including thefirst tag and the portion of the polypeptide. Each polynucleotide mayfurther include a second tag sequence. The promoter may be an induciblepromoter (e.g., a T7 promoter).

In a fourth aspect, the invention provides a method of determiningwhether a polypeptide is immunogenic which includes the steps of (a)individually contacting each member of the library of any of the aboveaspects with a second cell (e.g., a macrophage) capable of (i)endocytosing the cell or the virus in each member and (ii) displaying apeptide on its surface through the MHC class I pathway, where eachmember of the library includes the polypeptide encoded by thepolynucleotide, (b) individually contacting each member of step (a) witha CTL cell (e.g., a plurality of CTL cells) derived from a mammalpreviously infected with the pathogenic organism or a mammal having orhaving previously had a neoplasm; and (c) detecting whether the CTL cellis activated, where activation of the CTL cell determines whether thepolypeptide contained in the member is immunogenic. Each member of thelibrary may include a polynucleotide encoding a pore-forming protein(e.g., LLO). Each member of the library may be killed prior to thecontacting step (a). Prior to the contacting step (b), the second cellmay be killed. The method may further include a step (d) recovering thepolynucleotide encoding the polypeptide identified in step (c) from areplica copy of the library or may include a step, prior to contactingstep (a), making a replica of the library. The method may furtherinclude performing the method steps (b) and (c) at least one (e.g., 2,3, 4, 5, 7, 10, or 15) further time or times using the library, whichmay involve using a different CTL (e.g., a plurality of CTL cells) eachtime steps (b) and (c) are performed. In another embodiment, the methodmay include step (d) identifying an epitope sufficient for CTLactivation within the polypeptide determined to be immunogenic in step(c).

In a fifth aspect, the invention also provides compositions (e.g.,pharmaceutical compositions or vaccines) including at least one (e.g.,2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 25, 30, 40, or 50) epitope orpolypeptide identified using the fourth aspect of the invention. Thecompositions may further include a pharmaceutically acceptable carrier.

In a sixth aspect, the invention features compositions and methodsrelated to the discovery of the CT788 polypeptide (SEQ ID NO:1) as animmunogenic protein in Chlamydia infection and identification ofCT788₁₃₃₋₁₅₂ (SEQ ID NO:2) as antigenic epitope. Based on thisdiscovery, the invention features a purified or recombinant polypeptideincluding CT788 polypeptide (e.g., a CT788 polypeptide or a fusionprotein including a CT788 polypeptide), a composition includingpolypeptide including a CT788 polypeptide (e.g., a CT788 polypeptide ora fusion protein including a CT788 polypeptide), and a purified orrecombinant polypeptide including a fragment of a CT788 polypeptide(e.g., where the fragment is immunogenic or a fusion protein of saidfragment) such as KGIDPQELWVWKKGMPNWEK (SEQ ID NO:2) or including afragment having an amino acid sequence selected from the groupconsisting of the sequences listed in Table 1 (e.g., an immunogenicfragment listed in Table 1). Also featured is a composition including apolypeptide including a fragment of a CT788 polypeptide (e.g., animmunogenic fragment) such as KGIDPQELWVWKKGMPNWEK (SEQ ID NO:2) orincluding a fragment having an amino acid sequence selected from thegroup consisting of the sequences listed in Table 1 (e.g., animmunogenic fragment listed in Table 1). The CT788 polypeptide orfragment thereof may contain at least 1, 2, 3, 4, 5, 8, 10, 15 or moreadditional amino acids at either the N-terminal or C-terminus of themolecule.

TABLE 1  Fragments of Cta1₁₃₃₋₁₅₂ (including SEQ ID NOS: 3-154) KGI GMPWKKG VWKKG WVWKKG WVWKKGM VWKKGMPN GID MPN KKGM WKKGM VWKKGM VWKKGMPWKKGMPNW IDP PNW KGMP KKGMP WKKGMP WKKGMPN KKGMPNWE DPQ NWE GMPN KGMPNKKGMPN KKGMPNW KGMPNWEK PQE WEK MPNW GMPNW KGMPNW KGMPNWE KGIDPQELW QELKGID PNWE MPNWE GMPNWE GMPNWEK GIDPQELWV ELW GIDP NWEK PNWEK MPNWEKKGIDPQEL IDPQELWVW LWV IDPQ KGIDP KGIDPQ KGIDPQE GIDPQELW DPQELWVWK WVWDPQE GIDPQ GIDPQE GIDPQEL IDPQELWV PQELWVWKK VWK PQEL IDPQE IDPQELIDPQELW DPQELWVW QELWVWKKG WKK QELW DPQEL DPQELW DPQELWV PQELWVWKELWVWKKGM KKG ELWV PQELW PQELWV PQELWVW QELWVWKK LWVWKKGMP KGM LWVWQELWV QELWVW QELWVWK ELWVWKKG WVWKKGMPN WVWK ELWVW ELWVWK ELWVWKKLWVWKKGM VWKKGMPNW VWKK LWVWK LWVWKK LWVWKKG WVWKKGMP WKKGMPNWE WVWKKKKGMPNWEK KGIDPQELWV VWKKGMPNWE WVWKKGMPNWE LWVWKKGMPNWE GIDPQELWVWWKKGMPNWEK VWKKGMPNWEK WVWKKGMPNWEK IDPQELWVWK KGIDPQELWVW KGIDPQELWVWKKGIDPQELWVWKK DPQELWVWKK GIDPQELWVWK GIDPQELWVWKK GIDPQELWVWKKGPQELWVWKKG IDPQELWVWKK IDPQELWVWKKG IDPQELWVWKKGM QELWVWKKGM DPQELWVWKKGDPQELWVWKKGM DPQELWVWKKGMP ELWVWKKGMP PQELWVWKKGM PQELWVWKKGMPPQELWVWKKGMPN LWVWKKGMPN QELWVWKKGMP QELWVWKKGMPN QELWVWKKGMPNWWVWKKGMPNW ELWVWKKGMPN ELWVWKKGMPNW ELWVWKKGMPNWE LWVWKKGMPNWLWVWKKGMPNWEK KGIDPQELWVWKKG KGIDPQELWVWKKGMP GIDPQELWVWKKGMGIDPQELWVWKKGMPN IDPQELWVWKKGMP IDPQELWVWKKGMPNW DPQELWVWKKGMPNDPQELWVWKKGMPNWE PQELWVWKKGMPNW PQELWVWKKGMPNWEK QELWVWKKGMPNWEKGIDPQELWVWKKGMPN ELWVWKKGMPNWEK GIDPQELWVWKKGMPNW KGIDPQELWVWKKGMIDPQELWVWKKGMPNWE GIDPQELWVWKKGMP DPQELWVWKKGMPNWEK IDPQELWVWKKGMPNKGIDPQELWVWKKGMPNW DPQELWVWKKGMPNW GIDPQELWVWKKGMPNWE PQELWVWKKGMPNWEIDPQELWVWKKGMPNWEK QELWVWKKGMPNWEK KGIDPQELWVWKKGMPNWEGIDPQELWVWKKGMPNWEK

The invention also features pharmaceutical and vaccine compositions. Inone embodiment, the invention features a pharmaceutical compositionincluding a polypeptide including a CT788 polypeptide or including afragment of a CT788 polypeptide (e.g., an immunogenic fragment such asthose described above) and a pharmaceutically acceptable carrier. Thepharmaceutical composition may be formulated for administration by anymeans known in the art such as those described herein (e.g., oral orparenteral). In another embodiment, the invention features a vaccineincluding a polypeptide including a CT788 polypeptide or including afragment of a CT788 polypeptide (e.g., an immunogenic fragment such asthose described above), and a pharmaceutically acceptable carrier. Thevaccine may further include an adjuvant (e.g., those described herein)and may be formulated for administration by any means known in the artsuch as those described herein (e.g., oral or parenteral). In any of theabove embodiments of the sixth aspect, the polypeptide may have thesequence of a CT788 polypeptide or the sequence of a CT788 fragment(e.g., those shown in Table 1 and in Table 2). A polypeptide of theinvention may be a fusion protein. Fusion proteins may include a taguseful in purification, e.g., GST, His, or myc, or may include anprotein from an immune molecule (e.g., an Ig protein such as IgG, IgM,IgA, or IgE or an Fc region of an Ig protein), or any tag describedherein or known in the art.

The invention also features methods for treating or preventing aninfection (e.g., a bacterial infection such as a Chlamydia infection) inan individual (e.g., an individual in need of such treatment). Themethod includes administering (e.g., in an amount sufficient to preventor treat said bacterial infection) to an individual a CT788 polypeptideor fragment thereof (e.g., an immunogenic fragment such as thosedescribed above). The administered CT788 polypeptide or fragment thereofmay be purified polypeptide or fragment thereof.

By “portion” or “fragment” of a polypeptide is meant at least 5, 6, 7,8, 9, 10, 15 20, 30, 50, 100, 200, 300, or 500 amino acids of thepolypeptide. A fragment or portion may include 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, or even 100% of the full lengthpolypeptide (e.g., the polypeptide coded for by the genome of anorganism).

By “operably linked” is meant that a nucleic acid molecule and one ormore regulatory sequences (e.g., a promoter) are connected in such a wayas to permit expression and/or secretion of the product (i.e., apolypeptide) of the nucleic acid molecule when the appropriate molecules(e.g., transcriptional activator proteins) are bound to the regulatorysequences.

By “polypeptide encoded by the genome of a pathogenic organism” is meanta polypeptide having at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99%, or even 100% sequence identity to the polypeptide encoded bythe pathogenic organism.

By “pathogenic organism” is meant any organism capable of infecting amammal. Exemplary pathogenic organisms are bacteria, viruses, protozoa,and fungi and include the organisms described herein. In the context ofthe present invention, “pathogenic organism” additionally refers to anorganism other than a cell or virus included in a library of theinvention.

By “pore-forming protein” is meant any polypeptide, when contacted to alipid bilayer membrane, which is capable of forming a channel or pore inthe membrane. The pore or channel can allow passage of a molecule suchas an ion, a polypeptide or a fragment thereof, or a polynucleotide, topass through the membrane.

By “LLO” is meant listeriolysin O, or any fragment or variant thereof(e.g., a polypeptide substantially identical to the LLO sequence)capable of forming a pore in a eukaryotic membrane.

By “a cell capable of endocytosing” is a cell meant having the abilityto internalize a particle such as a cell, virus, or macromolecule into amembrane-bound compartment.

By “differentially expressed” meant an increase in expression of atleast 5%, 10%, 25%, 50%, 75%, 100%, 150%, 250%, 500%, or 1000% ofexpression in a test cell (e.g., a neoplastic cell) as compared to acorresponding control cell.

By “CT788 polypeptide” is meant a polypeptide with at least 40%, 50%,60%, 70%, 80%, 90%, 95%, 99%, or even 100% identity to the sequenceshown in FIG. 14, and having the ability to stimulate an immune responsein an organism previously infected with Chlamydia.

By “tag sequence” is meant any sequence used to form of fusion protein(e.g., including a fragment or portion of a polypeptide describedherein). Exemplary tags include FLAG, His (e.g., His6), hemagglutinin(HA), Myc, glutathione S-transferase (GST), or any other tag known inthe art. A tag sequence may be present at either the N- or C-terminus ofthe protein.

By a “purified polypeptide” or “isolated polypeptide” is meant apolypeptide that has been separated from components which naturallyaccompany it. Typically, the polypeptide is substantially pure when itis at least 30%, by weight, free from the proteins andnaturally-occurring organic molecules with which it is naturallyassociated. In certain embodiments, the preparation is at least 50%,60%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% by weight, free frommolecules with which it is naturally associated. A purified polypeptidemay be obtained, for example, by extraction from a natural source; byexpression of a recombinant polynucleotide encoding such a polypeptide;or by chemically synthesizing the polypeptide. Purity can be measured byany appropriate method, for example, column chromatography,polyacrylamide gel electrophoresis, or by HPLC analysis.

By “substantially identical” is meant a polypeptide or nucleic acidexhibiting at least 50%, 75%, 85%, 90%, 95%, or even 99% identity to areference amino acid (e.g., CT788) or nucleic acid sequence. Forpolypeptides, the length of comparison sequences will generally be atleast 7 amino acids (e.g., 20 amino acids), preferably at least 30 aminoacids, more preferably at least 40 amino acids, and most preferably 50amino acids, or full-length. For nucleic acids, the length of comparisonsequences will generally be at least 20 nucleotides (e.g., 60nucleotides), preferably at least 90 nucleotides, and more preferably atleast 120 nucleotides, or full length.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Generally, stringent conditions are selected tobe about 5° C. to about 20° C., usually about 10° C. to about 15° C.,lower than the thermal melting point (T_(m)) for the specific sequenceat a defined ionic strength and pH. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of the target sequencehybridizes to a matched probe. Typically, stringent conditions will bethose in which the salt concentration is about 0.02 molar at pH 7 andthe temperature is at least about 60° C. For instance, in a standardSouthern hybridization procedure, stringent conditions will include aninitial wash in 6×SSC at 42° C. followed by one or more additionalwashes in 0.2×SSC at a temperature of at least about 55° C., typicallyabout 60° C. and often about 65° C.

Nucleotide sequences are also substantially identical for purposes ofthis invention when the polypeptides and/or proteins which they encodeare substantially identical. Thus, where one nucleic acid sequenceencodes essentially the same polypeptide as a second nucleic acidsequence, the two nucleic acid sequences are substantially identical,even if they would not hybridize under stringent conditions due todegeneracy permitted by the genetic code (see, Darnell et al. (1990)Molecular Cell Biology, Second Edition Scientific American Books W. H.Freeman and Company New York for an explanation of codon degeneracy andthe genetic code). Protein purity or homogeneity can be indicated by anumber of means well known in the art, such as polyacrylamide gelelectrophoresis of a protein sample, followed by visualization uponstaining. For certain purposes high resolution may be needed and HPLC ora similar means for purification may be utilized.

By “immunogenic” is meant a compound (e.g., a polypeptide or fragmentthereof) having the ability to stimulate an immune response in anorganism (e.g., an organism previously infected with Chlamydia).

Libraries with discrete members in defined locations provide severaladvantages over pooled libraries including increased sensitivity foreach individual antigen as well as a far more rapid screening procedure.

Other features and advantages of the invention will be apparent from thefollowing Detailed Description, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing conventional MHC class I antigenpresentation.

FIG. 2 is a schematic diagram showing stimulation of CD8⁺ effectorT-cell responses during Listeria monocytogenes infection.

FIG. 3 is a schematic diagram showing listeriolysin O (LLO)-mediatedescape of L. monocytogenes from a vacuole during infection.

FIG. 4 is a schematic diagram showing LLO-mediated delivery of apolypeptide expressed in E. coli to the cytosol of a cell capable ofendocytosing a bacterium.

FIG. 5 is a set of images showing delivery of E. coli expressing GFP andLLO to the cytoplasm of a cell.

FIG. 6 is a schematic diagram showing E. coli/LLO-mediated delivery ofantigens to the MHC class I pathway.

FIG. 7 is a schematic diagram of the modified Gateway system used incloning of the C. trachomatis library.

FIG. 8 is a schematic diagram showing the expression cloning strategyfor identification of antigens for any pathogen of interest.

FIG. 9 is a schematic diagram of the validation strategy of the libraryemployed using B3Z T-cells, which recognize the SIINFEKL tag (SEQ IDNO:155) present on the expressed proteins using the modified Gatewayvector described herein.

FIG. 10 is an image showing that library validation strategy works astheorized by successfully determining which proteins in the library wereexpressed.

FIG. 11 is a schematic diagram showing a strategy for screening forbreast cancer antigens.

FIG. 12 is an image showing a positive result from screening of the C.trachomatis library using a Chlamydia specific T-cell line.

FIG. 13A is a graph showing the results of CD4 and IFNγ expression fromflow cytometry using a CD4⁺ T-cell clone being mixed with eitheruninfected or Chlamydia-infected BMM cells. Results are gated on livecells.

FIG. 13B is a graph showing the number of Chlamydia IFUs detected 72hours after infection. Both previously infected (immune) mice and naivemice with the CD4⁺ T-cell clone identified herein show low levels ofinfection as compared to naive mice without the CD4⁺ T-cell clone.

FIG. 14 is the peptide sequence of the CT788 (Cta1) protein (SEQ IDNO:1) and CT788₁₃₃₋₁₅₂ (Cta1₁₃₃₋₁₅₂) (SEQ ID NO:2).

FIG. 15 is an image showing clones of E. coli expressing individual C.trachomatis ORFs cultured with BMMs and then incubated with the T cellclone NR9.2. This figure shows a plate where supernatant from thecorresponding assay wells was tested for IFNγ in an ELISA assay. E. coliexpressing Cta1 (encoded by ORF CT788) induced NR9.2 to secrete highlevels of IFNγ (>370 ng/ml) whereas E. coli expressing other Chlamydiaproteins in the library induced only background levels of IFNγ secretion(<26 ng/ml). The well indicated as Cta1 and ELISA Standards are yellow;other wells are colorless. The colorimetric intensity of the wells shownin the figure that do not correspond to Cta1 or the standards aretypical of results seen with all the other E. coli clones in thelibrary. ELISA standards corresponding to high amounts of IFNγ areindicated.

FIG. 16A is a plot showing CD4⁺ peripheral blood lymphocytes fromnon-transgenic and NR1 transgenic mice stained for the Vα2 and Vβ8.3 TCRelements expressed by T cell clone NR9.2. Results are gated on live CD4⁺cells.

FIG. 16B is a graph showing proliferation of splenocytes from NR1transgenic or non-transgenic mice in response to the indicatedconcentrations of Cta1₁₃₃₋₁₅₂ (SEQ ID NO:2). Proliferation was measuredby [³H]thymidine incorporation into cells. These results indicate thatNR1 TCR tg cells recognize Cta1₁₃₃₋₁₅₂ (SEQ ID NO:2).

FIG. 17 is a graph showing that the CD4⁺ T-cell clone (NR9.2) injectedinto naive mice proliferates following infection by Chlamydia.Proliferation of the CFSE-labeled TCR transgenic T-cells is observed asa shift in the transferred population to the left into an arbitrarilyset gate. CFSE-labeled NR1 or OTII cells were transferred into C57BL/6recipients. One day later, mice were infected intravenously with theindicated pathogen. Spleens were harvested three days later. Resultswere gated on live CD4⁺ Vα2⁺ cells to detect the NR1 TCR tg cells.

FIG. 18A is a set of plots showing CD69 and CD44 are upregulated andCD62L is downregulated on proliferating transgenic T cells in thedraining lymph nodes. Proliferation (reflected as a shift in thepopulation to the left) of CFSE-labeled CD4⁺ TCR transgenic cellstransferred into naive mice was measured 5-7 days after infection of therecipient mice with C. trachomatis serovar L2. CD69 was upregulated onproliferating cells as reflected in a population shift from the bottomof the graph to the top. CD62L was downregulated on proliferating cellsas reflected in a population shift from the top of the graph to thebottom. Results are gated on live Thy1.2⁺CD4⁺ cells.

FIG. 18B is a pair of graphs showing transferred TCR transgenic cellsbegin proliferating extensively at four days post-infection in the lymphnodes draining the genital tract, but not in lymph notes draining othersites. Dotted line represents uninfected mice; the solid line representsinfected mice. Results are gated on live Thy1.2⁺(CD90.2)⁺CD4⁺ cells.

FIG. 18C is a set of plots showing transferred TCR transgenic T cellsare recruited to the genital tracts of mice following intrauterineinfection with Chlamydia. CFSE-labeled NR1 cells were transferred intoCD90.1 recipients and then mock-infected or infected in the uterus with106 IFU of C. trachomatis serovar L2. Seven days after infection, thegenital tracts were removed from the mice and analyzed for the presenceof the transferred NR1 cells. The presence of CD90.2⁺ CD4⁺ NR1 cells wascompared in the genital tracts of mock infected and infected recipients.Results were gated on live cells. The box shows adoptively transferred Tcells in the genital tissue of mice.

FIG. 19 is a set of graphs showing that NR1 cells proliferatepreferentially in the ILNs following intrauterine infection with C.trachomatis. CFSE-labeled NR1 cells were transferred into CD90.1recipients, which were then mock infected or infected in the uterus with10⁶ IFU of C. trachomatis serovar L2. ILNs and NDLNs were harvested atthe indicated times post-infection and proliferation of CD4⁺ NR1 cellswas examined. Results were gated on live CD90.2⁺ CD4⁺ Vα2⁺ cells tospecifically detect the NR1 TCR tg cells.

FIG. 20 is a set of graphs showing that NR1 cells differentiate into Th1cells. CFSE-labeled NR1 cells were transferred into CD90.1 recipientswhich were then infected in the uterus with 10⁶ IFU of C. trachomatisserovar L2. Six days later, cells from the ILNs were stimulated withPMA/ionomycin and examined for intracellular IFNγ by flow cytometry.Cta1-specific T cells (CD90.2⁺CD4⁺) were gated on CFSE^(lo) andCFSE^(hi) cells, and intracellular IFNγ levels in these two populationswere compared. Solid lines represent isotype control; gray filledhistograms indicate IFNγ.

FIG. 21A is a set of graphs showing the levels of CD62L, CD44, and CFSEon NR1 cells in the genital tracts of mock infected and infected mice.Results were gated on live CD90.2⁺ CD4⁺ cells to specifically detect theNR1 TCR tg cells.

FIG. 21B is a graph showing NR1 cells from the genital tract stimulatedwith PMA/ionomycin and analyzed by flow cytometry to detect productionof IFNγ. Results are gated on live CD90.2⁺ CD4⁺ cells to detect the NR1TCR tg cells specifically. The solid line represents the isotypecontrol; the filled histogram represents IFNγ.

FIG. 22 is a graph showing T cell clone NR9.2 recognizes the novel Tcell antigen Cta1₁₃₃₋₁₅₂ (SEQ ID NO:2). The antigenic peptide from Cta1was mapped by testing overlapping 20-mer synthetic peptides for theirability to stimulate NR9.2 to secrete IFNγ. Only Cta1₁₃₃₋₁₅₂ stimulatedNR9.2 to secrete significant levels of IFNγ in an IFNγ ELISA. Shownbelow is the protein sequence of Cta1 (SEQ ID NO:1) with Cta1₁₃₃₋₁₅₂(SEQ ID NO:2) underlined.

DETAILED DESCRIPTION

In one aspect, the invention permits in vitro screening of proven humanimmunity effectors to identify their key target antigens from thecomplete proteome, or a portion thereof, from any disease-causing agentand from differentially expressed polypeptides in neoplastic cells. Inanother aspect, the invention provides compositions, including purifiedproteins and vaccines, and methods of treating or preventing a Chlamydiainfection involving use of the CT788 polypeptide or fragment thereof asan antigen.

The technology of the first aspect of the invention enables a researcherto predict which epitope cocktail will prove effective in vivo, eitheras a prophylactic or a therapeutic vaccine. Importantly, it mimics themammalian immune system in vitro and presents it with every antigen thata given disease-causing agent might express in the infected host. Withina matter of a few days, it is possible to identify, from the entireproteome of a disease-causing agent, or portion thereof, the specificantigens that will stimulate the immune system most effectively in vivo,a task that previously was impossible.

At the core of the invention is the ability to rapidly identify antigensthat result in the in vivo stimulation of protective cytotoxicT-lymphocytes, allowing identified immune targets to be incorporatedimmediately into existing antigen delivery systems to producemultivalent vaccine formulations with the highest probability ofgenerating protective cell-mediated immunity.

One of the key components of a protective cell-mediated immune responseare CD8⁺ cytotoxic T-lymphocytes (CTLs), which can recognize andeliminate pathogen-infected host cells, preventing dissemination of thepathogen within the host. The generation of CTLs during a naturalinfection often requires the production of pathogen-specific antigenicproteins within the cytosol of host cells. During intracellularinfection, antigenic proteins present within the host cell cytosol areproteolytically degraded into peptides. These peptides are subsequentlydisplayed on the surface of host cells in association with majorhistocompatibility complex (MHC) class I molecules (FIGS. 1 and 2).Peptide/MHC complexes on the surface of host cells are recognized byCTLs, leading to CTL-mediated killing of infected host cells and thedevelopment of protective T-cell memory. However, relatively fewadvances have been made in developing strategies to identifypathogen-specific antigens efficiently that are used as targets for theMHC class I pathway. Such targets are the frontline materials forincorporation into component vaccines to stimulate protective CTL memoryand prevent infection by an invading microorganism. The development ofeffective methods to identify pathogen-specific antigenic proteins andtarget them to the MHC class I presentation pathway is therefore offundamental importance in the rational design of vaccines againstintracellular pathogens. While several current vaccine strategies are indevelopment for in vivo antigen delivery, prior to the presentinvention, no strategy existed for the rapid determination of the entireantigenic profile of an infectious disease pathogen to determine themost appropriate antigenic determinants to include in a vaccineformulation.

The present invention allows for the efficient in vitro expression ofthe entire protein complement of an infectious pathogen coupled withtargeting of the expressed proteins to host antigen-presenting cells(APC) for the generation of CTL responses. Antigens delivered to hostcells through this targeted expression system are processed by the MHCclass I pathway (FIG. 1) for presentation to CTLs. Candidate vaccineantigens are identified in this manner through the use ofpathogen-specific CTL lines that have been generated from previouslyinfected individuals.

As outlined below, the present invention has been applied to Chlamydiatrachomatis, an intracellular bacterial pathogen and the most commonsexually transmitted disease agent in the United States. C. trachomatisis also the leading cause of preventable blindness worldwide, and novaccine is currently available. C. trachomatis-specific CTL lines thatprovide protective immunity in adoptive transfer studies were obtainedfrom a mouse model and used to identify CTL-eliciting antigens. Oneantigen that corresponds to a unique member of the 894 possible C.trachomatis open reading frames, was identified from each CTL linescreened against a C. trachomatis expression library. The identifiedantigens stimulate protective CTLs during C. trachomatis infection.Methods for producing libraries of the invention and performing thescreening assays are described herein.

Generation of Libraries from a Pathogenic Organism

To generate a library of the invention, open reading frames or portionsthereof from the genome of a pathogenic organism (e.g., a virus or abacterium) are cloned into vectors capable of being expressed (e.g.,operably linked to a promoter) in the cell or virus of which the membersof the library include. This may be accomplished by any means known inthe art. In one embodiment, PCR primers are designed to amplify openreading frames identified in the genome of a pathogenic organism. Setsof primers may be designed to amplify 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 98%, 99%, or even 100% of the open reading frames, orportions thereof, from the genome of the pathogenic organism. Primerdesign may be performed using a computer program, for example, the GAP(Genome-wide Automated Primer finder server) computer program, availablefrom the University of California at Irvine, which allows rapid designof primers targeting large numbers of open reading frames from thegenome of a pathogen. In one embodiment, primers are designed such thatsecretion signal sequences from the proteins of the pathogenic organismare removed. Pathogenic bacteria and viruses whose genomes have beensequenced or are being sequenced may be found, for example, at theGenome Online Database (GOLD) (Liolios K et al., Nucleic Acids Res.34(Database issue):D332-334, 2006). Pathogenic organisms (e.g.,bacterial pathogens) whose genomes are fully sequenced are particularlyuseful in the present invention.

Alternatively, reverse transcription of mRNA may be used to generate alibrary of the invention. Reverse transcriptase in conjunction with aprimer targeted to a specific mRNA sequence may be used to transcribeinto a DNA sequence the coding region, or portion thereof, containedwithin the mRNA. Subsequent amplification by PCR (e.g., as describedherein) may be used to generate sufficient quantities of DNA for thecloning the desired polynucleotide in to an expression vector.

To generate the discrete members of the library, each PCR reactioncontaining primers specific to an open reading frame or portion thereofmay be carried out in a separate reaction volume (e.g., in an 96 wellplate). In one embodiment, a two step PCR process is used. The first setof primers is used to amplify the desired open reading frames, and asecond set of primers is used to append additional sequences onto theamplified sequences for cloning. Such additional sequences may includesites for restriction enzymes or recombination sequences for cloning(e.g., the Gateway system from Invitrogen or the MAGIC system (Li et al.Nat. Genet. 7(3):311-9, 2005)), or any sequence tag known in the art(e.g., a His₆ tag, a myc tag, or a SIINFEKL epitope (SEQ ID NO:155)).The PCR products are introduced into the cloning vectors as appropriatefor the system used, as is known in the art. If a cloning vector lacks apromoter capable of driving expression in the cell or virus of thelibrary, it will be subsequently necessary to clone the ORF or fragmentthereof into a vector containing a promoter. During any of the clonesteps, peptide tags, such as those described herein, may be added toeither the N-terminus or C-terminus of the polypeptides clones, orportions thereof.

Once the polynucleotide encoding at least portions of the polypeptidesare cloned into vectors capable of expression, they may each beintroduced in to an appropriate host cell or virus, thereby forming alibrary of the invention. The methods described herein have been used tocreate a library expressing 888 of the 894 polypeptides found in the C.trachomatis genome.

Pathogenic organisms useful in the invention include, for example,adenovirus, Ascaris lumbricoides, astrovirus, Bacteroides spp.,beta-hemolytic streptococci, BK virus, Blastocystis hominis, Blastomycesdermatitidis, Bordetella pertussis, Bunyavirus, Campylobacter fecalis,Candida spp., Chlamydia pneumoniae, Chlamydia psittaci, Chlamydiatrachomatis, Clonorchis sinensis, Clostridium difficile, Clostridiumspp., Coagulase-negative staphylococci, Coccidoides immitis,Cornybacterium diphtheriae, Cornybacterium spp., coronavirus,coxsakievirus A, coxsakievirus B, Cryptococcus neoformans,cryptosporidium, cytomegalovirus, echovirus, Entamoeba histolytica,Enterbater spp., Enterobius vermicularis, Enterococcus spp.,Epstein-Barr virus, equine encephalitis virus, Escherichia coli,Escherichia spp., fungi, Giardia lamblia, Haemophilus influenzae,hepatitis virus, hepatitis C virus, herpes simplex virus, Histoplasmacapsulatum, HIV, Hymenolepis nana, influenza virus, JC virus, Klebsiellaspp., Legionella spp., Leishmania donovani, lymphocytic choriomeningitisvirus, microfilariae, microsporidium, Mycobacterium tuberculosis,Mycoplasma pneumoniae, myxovirus, Necator americanus, Nocardia spp.,Norwalk virus, Opisthorchis viverrini, parainfluenza virus,paramyxovirus, Plasmodium spp., Pneumocystis carinii, Proteus spp.,Pseudomonas aeruginosa, Pseudomonas spp., rabies virus, respiratorysyncytial virus, rhinovirus, rotavirus, Salmonella, Shigella, St. Louisencephalitis virus, Staphylococcus aureus, Streptococcus pneumoniae,Strongyloides stercoralis, togavirus, Toxoplasma spp., Trichuristrichiura, Varicella-Zoster virus, vibrio cholera, Viridansstreptococci, and Yersinia enterocolitica.

Particularly useful the present invention are obligate intracellularpathogens. Intracellular bacteria include, for example, Anaplasma bovis,A. caudatum, A. centrale, A. marginale A. ovis, A. phagocytophila, A.platys, Bartonella bacilliformis, B. clarridgeiae, B. elizabethae, B.henselae, B. henselae phage, B. quintana, B. taylorii, B. vinsonii,Borrelia afzelii, B. andersonii, B. anserina, B. bissettii, B.burgdorferi, B. crocidurae, B. garinii, B. hermsii, B. japonica, B.miyamotoi, B. parkeri, B. recurrentis, B. turdi, B. turicatae, B.valaisiana, Brucella abortus, B. melitensis, Chlamydia pneumoniae, C.psittaci, C. trachomatis, Cowdria ruminantium, Coxiella burnetii,Ehrlichia canis, E. chaffeensis, E. equi, E. ewingii, E. muris, E.phagocytophila, E. platys, E. risticii, E. ruminantium, E. sennetsu,Haemobartonella canis, H. felis, H. muris, Mycoplasma arthriditis, M.buccale, M. faucium, M. fermentans, M. genitalium, M. hominis, M.laidlawii, M. lipophilum, M. orale, M. penetrans, M. pirum, M.pneumoniae, M. salivarium, M. spermatophilum, Rickettsia australis, R.conorii, R. felis, R. helvetica, R. japonica, R. massiliae, R.montanensis, R. peacockii, R. prowazekii, R. rhipicephali, R.rickettsii, R. sibirica, and R. typhi. Exemplary intracellularprotozoans are Brachiola vesicularum, B. connori, Encephalitozooncuniculi, E. hellem, E. intestinalis, Enterocytozoon bieneusi,Leishmania aethiopica, L. amazonensis, L. braziliensis, L. chagasi, L.donovani, L. donovani chagasi, L. donovani donovani, L. donovaniinfantum, L. enriettii, L. guyanensis, L. infantum, L. major, L.mexicana, L. panamensis, L. peruviana, L. pifanoi, L. tarentolae, L.tropica, Microsporidium ceylonensis, M. africanum, Nosema connori,Nosema ocularum, N. algerae, Plasmodium berghei, P. brasilianum, P.chabaudi, P. chabaudi adami, P. chabaudi chabaudi, P. cynomolgi, P.falciparum, P. fragile, P. gallinaceum, P. knowlesi, P. lophurae, P.malariae, P. ovale, P. reichenowi, P. simiovale, P. simium, P.vinckeipetteri, P. vinckei vinckei, P. vivax, P. yoelii, P. yoeliinigeriensis, P. yoelii yoelii, Pleistophora anguillarum, P.hippoglossoideos, P. mirandellae, P. ovariae, P. typicalis, Septataintestinalis, Toxoplasma gondii, Trachipleistophora hominis, T.anthropophthera, Vittaforma corneae, Trypanosoma avium, T. brucei, T.brucei brucei, T. brucei gambiense, T. brucei rhodesiense, T. cobitis,T. congolense, T. cruzi, T. cyclops, T. equiperdum, T. evansi, T.dionisii, T godfreyi, T. grayi, T. lewisi, T. mega, T. microti, T.pestanai, T. rangeli, T. rotatorium, T. simiae, T. theileri, T. varani,T. vespertilionis, and T. vivax.

Infectious fungi that may be used in the invention include yeast such asCandida albicans, Candida stellatoidea, Candida tropicalis, Candidaparapsilosis, Candida krusei, Candida pseudotropicalis, Candidaquillermondii, Candida glabrata, Candida lusianiae, and Candida rugosa.Other fungi include Microsporum canis and other M. spp., Trichophytonspp. (e.g., T. rubrum and T. mentagrophytes), Torulopsis glabrata,Epidermophyton floccosum, Malassezia furfur, Pityropsporon orbiculare,P. ovale, Cryptococcus neoformans, Aspergillus fumigatus and otherAspergillus spp., Zygomycetes (e.g., Rhizopus, Mucor), Paracoccidioidesbrasiliensis, Blastomyces dermatitides, Histoplasma capsulatum,Coccidioides immitis, and Sporothrix schenckii.

For generation of a library in a bacterial host, any vector capable ofexpressing the cloned polynucleotide may be used in the host bacteria.In one embodiment, a laboratory strain of E. coli is used; appropriatevectors for expression in E. coli are well known in the art and includevectors such as the pET expression vector system (EMD Biosciences, SanDiego, Calif.), pDESTSL8 (described herein), and pDEST17 (Invitrogen).Vectors may be modified to include N- or C-terminal tags, as desired,for example, for use in verification of the library (e.g., as describedherein). Such vectors may also contain an inducible promoter (e.g., theT7 polymerase promoter) as are well known in the art, which allow forexpression upon application of an exogenous chemical (e.g., IPTG(isopropyl-beta-D-thiogalactopyranoside)) or upon application of a phage(e.g., CE6 phage) depending on the bacterial strain used.

For generation of viral libraries (e.g., phage display libraries),polynucleotides forming a library are cloned into phage vectors. Suchvectors and vector systems are well known in the art and include theNovagen T7Select® phage display vectors (EMD Biosciences).

In certain embodiments, bacterial libraries of the invention, inaddition to containing a first polynucleotide from the pathogenicorganism, may contain an second polynucleotide sequence, either as partof the first polynucleotide (in addition to the sequence from thepathogenic organism) or as part of a second expression vector. Thissecond polynucleotide sequence may encode a second protein, for example,listeriolysin O (LLO).

E. coli/LLO System

The E. coli/LLO system provides a means for a protein expressed in E.coli, following endocytosis into a cell, to escape from the vacuole inwhich it is endocytosed, and contact the cytosol of the cell (FIG. 3).LLO acts by perforating the vacuole, thereby allowing its contents toescape into the cytoplasm. Listeriolysin O exhibits greater pore-formingability at mildly acidic pH (the pH conditions within the vacuole) andis therefore well suited for this purpose. This allows for enhancedprocessing of the expressed protein through the MHC class I pathway (seeFIGS. 4-6). This system is described extensively in U.S. Pat. No.6,004,815, which is hereby incorporated by reference. In addition toLLO, the libraries and methods of the invention may employ otherproteins with similar activity; any protein (e.g., a pore-formingprotein) that facilitates delivery of potentially antigenic proteins tothe cytoplasm of a cell capable of endocytosing the bacteria or virus ofthe library of the invention may be employed. The E. coli/LLO system isparticularly useful for screening for antigens that activate CD8⁺ CTLcells. Libraries of the invention prepared without LLO may be used toscreen for CD4⁺ CTL cell activation.

The examples presented herein are intended to illustrate, rather thanlimit, the present invention.

EXAMPLE 1 Cloning Chlamydia trachomatis Genome

Each ORF in the Chlamydia trachomatis serovar D/UW-3/Cx genome wasamplified through a 2-step PCR procedure. The first step used primersspecific for each ORF to amplify each sequence in a 96-well format. Theprimers were designed to remove any secretion signal sequences presentin the ORFs to prevent secretion in E. coli when they are expressed andto minimize toxicity. The second PCR step was used to add the requiredrecombination sequences to the 5′ and 3′ ends of each PCR product.

Once the ORFs were amplified and the recombination sequences were added,the PCR products were recombined into a DONR vector. This can be doneusing the Gateway system or the MAGIC system. For this library, theGateway system was used. The final PCR product was incubated with thepDONR221 plasmid in the presence of Gateway BP recombinase. After anovernight incubation, the reaction solution was transformed directlyinto E. coli which were then plated on kanamycin-containing LB agarplates to select for the presence of the DONR plasmid. Any bacteria thatgrow must contain a DONR vector which has recombined with a PCR productbecause the DONR plasmid is toxic to E. coli if it has not recombineddue to the presence of a toxin gene in the plasmid. When the DONRplasmid recombines with a PCR product this toxin gene is lost allowingthe bacteria to support the presence of the plasmid.

The MAGIC recombination system works very similarly, except the PCRproduct is transformed directly into E. coli which already contain theMAGIC1 donor vector. The recombination step occurs within the bacteria.Successful recombination events are then selected for by plating thebacteria on plates containing chlorophenylalanine. This chemical istoxic to E. coli containing the magic donor vector that has notrecombined with the PCR product due to the presence of the pheS gene inthe vector, which again is lost during a successful recombination. OnlyE. coli that contain a donor vector that has recombined with the PCRproduct survive. The whole transformation process in either system isdone in a 96-well format including the plating procedure to ensure thatall colonies appearing in any well on the agar plate are the result of asuccessful recombination of only the single ORF sequence that wasamplified in the corresponding well during the PCR step. This allows forthe cloning of each ORF in clonal populations.

A clone for each ORF was inoculated into LB containing kanamycin in96-deep well plates. The plasmid DNA from each clone was isolatedthrough a 96-well mini-prep procedure. Primers complimentary tosequences in the DONR vector 5′ and 3′ of the cloned ORF sequence wereused to PCR amplify the sequence that was recombined into the vector.The PCR product was run on an agarose gel and the size of the productwas compared to the predicted size. Any clone that contained arecombined sequence which was significantly different from the predictedsize was abandoned and a new clone for that ORF was chosen and testedfor the proper size. All clones that contained sequences of the properlength were moved on to the next step of the cloning procedure.

For the MAGIC system, the donor vector can be used to express the ORFs;thus, no further cloning is required. In the Gateway system, however,the ORF sequence is shuttled to a second vector containing a promoterthat allows for expression of the ORF. This was accomplished byincubating the isolated DONR plasmid DNA with the destination vectorpDESTSL8 in the presence of Gateway LR recombinase. pDESTSL8 wasconstructed in our lab from pDEST17 (FIG. 7) by adding a C-terminalfusion which contains the SIINFEKL epitope (SEQ ID NO:155). After an 18hr incubation, the reaction solution was transformed directly into E.coli which was then plated on carbenicillin-containing LB agar plates ina 96-well format. A clone for each ORF was inoculated intocarbenicillin-containing LB in 96-well plates to select for the bacteriathat have taken up pDESTSL8 that has recombined in the ORF sequence. Theplasmid DNA for each clone was isolated and primers complimentary tosequences in pDESTSL8 5′ and 3′ of the recombined ORF were used to PCRamplify the recombined sequence. These product of the PCR amplificationwere analyzed by agarose gel electrophoresis; any clone with an insertof an incorrect size was abandoned and a new clone for that ORF wastested. Every clone with an insert with the correct size was deemedcorrect and was moved on to be tested for expression.

Generation of Libraries from Neoplastic Cells

In another embodiment of the invention, a replicable library thatincludes polynucleotides encoding at least fragments of polypeptideswhose expression is increased (e.g., by a factor of at least 1.05, 1.1,1.2, 1.4, 1.5, 1.75, 2, 3, 4, 5, 7, 10, 25, 50, or 100 times) in aneoplastic cell such as a cancer cell (e.g., a breast cancer cell) ascompared to the corresponding normal cell is provided. Identification ofa set of polynucleotides with increased expression in neoplastic cellsmay be performed by any method known in the art. Typically, expressionis profiled using an expression array, such as those available fromAffymetrix. Polynucleotides whose expression is increased in aneoplastic cell are thus identified using such expression arrays and maybe subsequently used to generate a library of the invention.

Cloning of polynucleotides whose expression is increased in a neoplasmmay be performed by reverse transcription of the individual mRNAstranscribed from each polynucleotide identified as having increasedexpression. Primers specific to each mRNA are selected and used toindividually transcribe the mRNA sequences into DNA sequences. The DNAsequences may then be cloned in an appropriate vector containing apromoter capable of expression in the cell or virus of the library,typically following amplification of each DNA sequence by PCR. Once eachpolynucleotide is cloned into a vector, the polynucleotides may beintroduced into a cell or virus as described herein.

Polynucleotides overexpressed in neoplastic cells as compared to normalcells may also be identified through the use of cDNA subtractionlibraries. Methods for generating such libraries are known in the artand are commercially available, for example, the Clonetech PCR-Selectproducts (Clonetech Laboratories, Inc., Mountain View, Calif.).

Expression of Polynucleotides

For use in the methods of the invention, a cell or virus forming amember of a library can express the polynucleotide encoding at least aportion of a polypeptide from the pathogenic organism. In bacterialsystems with inducible promoters, this is accomplished by administrationof the appropriate substance (e.g., chemical or phage) to induce proteinexpression. In the case of the C. trachomatis library described inExample 1, this was performed as follows.

EXAMPLE 2 Expression of C. trachomatis Polynucleotides

Each expression plasmid containing an ORF was transformed into E. coliwhich already contained a plasmid to express the cytosolic form oflisteriolysin O (cLLO). The bacteria were plated on LB agar platescontaining both carbenicillin and chloramphenicol to select for bothplasmids. A colony for each ORF was picked, inoculated intocarbenicillin and chloramphenicol containing LB in 96-well plates andgrown for 18 hrs. The stationary phase culture was then diluted intofresh LB containing 0.2% maltose, carbenicillin and chloramphenicol.After 4 hours of growth the OD₆₀₀ was taken for each well to determinethe number of bacteria in each well. MgSO₄ was added to bring theconcentration to 10 mM in each culture. CE6 phage, a replicationdeficient lambda phage which contains in its genome the gene for T7polymerase under a constitutive promoter, was then added at an MOI of12:1. Once the phage has infected the bacteria, the T7 polymerase isexpressed which can then transcribe a chlamydial ORF under the controlof a T7 promoter. The cultures were gently mixed and incubated withoutshaking for 20 minutes at 37° C. After 20 min, the cultures wereincubated shaking for an additional 1 hour and 40 minutes to allow forexpression of ORF. The OD₆₀₀ from each well was then measured todetermine the concentration of bacteria in each culture well. 1×10⁸bacteria were harvested from each well, pelleted and resuspended in 1mLof 0.5% paraformaldehyde. The cultures were incubated for 30 minutes atroom temperature. The cultures were then pelleted and washed three timeswith PBS. After the final wash, the cell were pelleted and resuspendedin 1 mL of RP-10 media. The cultures were then aliquoted out in volumesof 20 μL into 96-well plates and frozen at −80° C. This procedure canyield greater than 50 separate aliquots of the library to screendifferent T-cell lines.

Verification of the Expression of the Library

Any method known in the art may be used to determine whether each memberof the library is able to express the polynucleotide from the pathogenicorganism or a neoplastic cell (e.g., western blotting). In the C.trachomatis library described herein, verification of expression wasaccomplished as follows.

EXAMPLE 3 Testing for Expression

We developed a high-throughput test for protein expression utilizing theSIINFEKL epitope (SEQ ID NO:155) fused to the C-terminus of each ORF forverification of protein expression (FIGS. 8-10). To perform this assay,a frozen aliquot of the library is thawed and added to macrophages ofthe H2^(b) haplotype, which were seeded the previous day in 96-wellplates. The macrophage/bacteria mixture is incubated for an hour duringwhich time the bacteria are phagocytosed by the macrophages. In thephagosome, the bacteria are lysed, releasing all of the proteins beingexpressed by the E. coli into the vacuole including the chlamydialprotein and cLLO. The cLLO then perforates the phagosome membrane,allowing the chlamdyial protein access to the cytosol of the macrophagewhere it can be processed and peptides from the protein can be presentedon MHC Class I molecules. If the chlamydial protein was expressed tofull length, it has the SIINFEKL epitope (SEQ ID NO:155) fused to itsC-terminus. This epitope will be delivered along with the chlamydialprotein and will be processed and presented on the MHC Class I moleculeson the surface of the macrophages. This MHC-peptide complex is probedfor by adding B3Z T-cell hybridoma cells at the end of the hourincubation. B3Z cells become activated when they recognize theMHC-SIINFEKL complex (FIG. 9). When they become activated,β-galactosidase expression is induced. The B3Z cells are incubated withthe macrophages for 15-20 hours to allow the B3Z cells time to scan themacrophages for the MHC-peptide complex and upregulate β-galactosidaseif the complex is present. After 15-20 hours, LacZ buffer is added todetect the amount of β-galactosidase activity in each well of the plate.LacZ buffer contains a detergent to lyse the macrophages and aβ-galactosidase substrate, chlorophenyl red-β-D-galactopyranoside(CPRG), which turns from yellow to purple when it is cleaved byβ-galactosidase. The amount of cleaved product is determined bymeasuring the OD_(570 nm) of each well in a spectrophotometer. Thus, astrong signal at 570 nm indicates both that the chlamydial protein thatwas expressed in that well was expressed to full length and wasdelivered to the MHC Class I pathway.

Determining whether a Polypeptide from a Pathogen or Neoplastic Cell isImmunogenic

A library of cells or viruses contain polynucleotides encodingpolypeptides from a pathogenic organism or from a neoplastic cell may bescreened to determine which of the polypeptides encoded by thepolynucleotides are immunogenic. This may be accomplished by contactingeach member of the library with a second cell (e.g., a macrophage)capable of endocytosing the cell or virus of the library, and displayingportions of the expressed polypeptide of the library on the surface ofthe second cell. This process is described, for example, in U.S. Pat.No. 6,008,415. The second cell is then contacted with a CTL cell from anorganism previously infected with the pathogenic organism, a CTL cellfrom an organism with a neoplasm, or a CTL cell from an organism thatpreviously had a neoplasm. Contacting with a CTL cell may be proceededby fixing the second cell, e.g., using paraformaldehyde. A CTL capableof binding a presented portion of the antigen/protein will result insecretion of cytokines. Cytokine secretion (e.g., secretion of IFNγ,IL-2, or TNF) may be assayed for as is known in the art, for example,using an ELISA assay. In a working example, the C. trachomatis librarydescribed herein was screened as described in Example 4 below.

Cytotoxic T Lymphocytes

Pools of CTL cells for use in the methods of the invention may bederived by any means known in the art. Typically, in screening forantigens to pathogenic organisms, CTL cells are prepared from a mammalpreviously infected with the pathogen. This preparation will contain CTLcells specific for antigens from the pathogen.

C. trachomatis-specific CTLs may be elicited from mice as follows. Amouse was injected i.p. with 10⁷ IFU of C. trachomatis. 14 days laterthe mouse was euthanized and the spleen was harvested. The spleen wasmashed through a 70 μm screen to create a single cell solution ofsplenocytes. The CD8⁺ T-cells were isolated from the splenocytes usingα-CD8 antibodies bound to MACS magnetic beads using MACS separationprotocols standard in the art (see, for example, MACS technologyavailable from Miltenyi Biotec Inc., Auburn, Calif.). The isolated CD8⁺cells were added to macrophages of the same haplotype which wereinfected with C. trachomatis 18 hours prior in a 24-well dish.Irradiated splenocytes from a naïve mouse were added as feeder cells inmedia containing IL-2. The cells were incubated for 10 days during whichtime the C. trachomatis-specific T-cells were stimulated by the infectedmacrophages and replicated. On day 10 the T-cells were stimulated againusing macrophages infected with C. trachomatis 18 hours prior andirradiated splenocytes. This procedure was repeated until sufficientamounts of T-cells were present to screen the entire library.

For preparation of CTL cells for use in screening for antigens toneoplastic cells, e.g., breast cancer cells, polyclonal CTL poolsspecific to breast cancer are generated using whole tumor RNA. The CTLpool is then verified using known tumor-associated antigens (FIG. 11).

CTL cells may be cloned from a human subject as described by, forexample, Hassell et al. (Immunology 79:513-519, 1993).

EXAMPLE 4 Screening for Unknown Antigens

A frozen aliquot of the library (10 96-well plates in total) is thawedand added to macrophages which were seeded the previous day in 1096-well plates. The plates are incubated for 2 hours, washed and fixedwith 1% paraformaldehyde to kill the macrophages and stabilize any MHCClass I-peptide interactions. The cells are then washed again to removethe paraformaldehyde. C. trachomatis-specific CD8⁺ T-cells of unknownspecificity are then added to each well. If the epitope the T-cellsrecognize is being presented in a given well, the T-cells will becomeactivated. T-cell activation is detected by measuring the amount of IFNγpresent in the supernatant of each well after 18-20 hours of cultureusing an IFNγ ELISA kit (see FIG. 12). This may also be accomplished bytesting for other cytokines released by activated T-cells such as IL-2.Any well which contains a much higher concentration of IFNγ than all therest of the wells suggests the corresponding chlamydial protein in thelibrary is a possible antigen. For any possible antigen, the process isrepeated to ensure that the hit is not a false positive. If the antigenactivates the T-cells in the secondary screen, the ORF is deemed anantigen and is moved on to epitope identification.

Epitope Identification

Once a polypeptide antigen is identified, it is often desirable toidentify the epitope within the polypeptide to which the CTL cell isresponding. This may be accomplished by recursively assaying eachportion of the polypeptide using cloning techniques known in the art, ormay be achieved using a system such as the Erase-a-Base kit (Promega).The truncated polypeptides are screened against CTL cells as describedherein to determine which portions of the polypeptide are required togenerate an immunogenic response.

In one embodiment, the specific peptide epitope recognized by the T-cellline is identified by making nested deletions of the full antigensequence using the Erase-a-Base kit from Promega. The plasmid clonecontaining the antigen sequence is first digested with NheI and AatII.The NheI cut creates the proper 5′ overhang near the 3′ end of the ORFto allow the nucleases in the Erase-a-Base kit to cleave 3′-5′ throughthe antigen sequence while the 3′ overhang of the AatII cut siteprevents the nuclease from digesting in the other direction and removingthe sequence for the drug resistance cassette. Once the plasmid is cutwith the restriction enzymes, the Erase-a-Base kit is used to make aseries of 3′ truncations in the sequence encoding the antigen byfollowing the protocol supplied with the kit. These truncations areligated and transformed into E. coli containing the plasmid to expresscLLO. Protein expression is then induced with CE6 phage as describedabove. When expressed, the truncations will create proteins that aremissing increasing amounts of their C-terminal peptide sequence. Thedifferent truncation clones are then rescreened with the original T-cellline using the procedure outlined above. The clones that no longeractivate the T-cell line have lost the sequence of the epitoperecognized by the T-cell line. The sequences of the largest truncationclone that no longer contains the epitope and the shortest truncationclone that still does contain the epitope are determined. The locationof the 3′ edge of the epitope resides in the peptide sequence theshortest positive clone contains and the largest negative clone doesnot. Overlapping 8-10mer peptides of this sequence are synthesized. Thepeptides are then pulsed onto macrophages and screened for their abilityto activate the T-cell line. The peptide that is capable of activatingthe T-cell line is deemed the epitope.

CT788

The invention also features the CT788 (Cta1) polypeptide and fragmentsthereof (e.g., immunogenic fragments including amino acids 133-152 ofthe CT788 protein (SEQ ID NO:2) and those listed in Table 1 (SEQ IDNOS:3-154) (e.g., an immunogenic fragment listed in Table 1)),pharmaceutical and vaccine compositions including the CT788 protein orfragments thereof (e.g., those described herein), and methods fortreating or preventing a bacteria infection (e.g., a Chlamydiainfection) by administration of CT788 or a fragment (e.g., animmunogenic fragment) thereof (e.g., a fragment described herein).

Identification of CT788 as an Antigenic Protein of C. trachomatis

Although pathogen-specific TCR tg T cells had been used previously inother infectious disease models (Butz et al., Immunity 8:167-75, 1998;Sano et al., J. Exp. Med. 194:173-80, 2001; Roman et al., J. Exp. Med.196:957-68, 2002; Coles et al., J. Immunol. 168:834-838, 2002; McSorleyet al., Immunity 16:365-377, 2002), the NR1 mice described below are thefirst TCR tg mice with T cells specific for a pathogen that infects thegenital tract. Previously, it had not been possible to examine T cellresponses to genital pathogens such as C. trachomatis because it wasdifficult to identify and track naïve T cells specific for genitalantigens in vivo. In particular, the inability to modify C. trachomatisgenetically to express a heterologous T cell epitope as well as the lackof a well-defined Chlamydia-specific CD4⁺ T cell antigen had made itdifficult to study T cell responses to this genital pathogen.

Because murine CD4⁺ T cell antigens recognized during Chlamydiainfection had not been previously defined, analysis of primaryChlamydia-specific T cell responses had been limited to examiningpolyclonal T cell responses to undefined antigens (Cain et al., Infect.Immunol. 63:1784-1789, 1995). In these previous experiments, theinvestigators could not differentiate true Chlamydia-specific T cellresponses from bystander T cell activation, which has been shown tocontribute to the overall response in a number of other infectiousdisease models (Yang et al., J. Immunol. 136:1186-1193, 1986; Tough etal., Immunol. Rev. 150:129-142, 1996). As described below, we identifieda CD4⁺ T cell antigen Cta1 (CT788), which allows examination of anantigen-specific T cell response stimulated during C. trachomatisinfection. This antigen was predicted to be a periplasmic proteinbecause of an N-terminal signal sequence, but its function is unknown(Stephens et al., Science 282:754-759, 1998). Cta1 is conserved in allof the C. trachomatis serovars that have been sequenced, including thosethat cause genital tract infection, ocular infection, and LGV. Cta1stimulated protective T cells following natural infection, suggestingthat this protein may play a significant role in immunity against C.trachomatis.

Another difficulty in studying the initial encounter of T cells withantigen is the low precursor frequency of pathogen-specific T cells inan unimmunized animal. Other investigators have attempted to increasethe frequency of Chlamydia-specific T cells by transferring T cellclones of unknown specificity into Chlamydia-infected mice (Hawkins etal., Infect. Immunol. 68:5587-5594, 2000; Hawkins et al., Infect.Immunol. 70:5132-5139, 2002). Because the transferred T cells areantigen-experienced and have been propagated through multiple rounds ofrestimulation, the response of these T cells cannot be used to model theinitial encounter of naïve T cells with C. trachomatis. To overcome thelimitations of previous approaches and study the initialChlamydia-specific T cell response, we developed NR1, a TCR tg mouseline specific for Cta1. Here, adoptive transfer of NR1 cells intounimmunized mice was used to increase the frequency of naïve,Chlamydia-specific T cells while still maintaining the polyclonal T cellenvironment in the recipient animals (Pape et al., Immunol. Rev.156:67-78, 1997).

The response of Chlamydia-specific T cells to the infected murinegenital tract was then examined. An infection model where the uterus ofthe mouse was inoculated with the human C. trachomatis serovar L2 wasemployed. This route of infection had been previously used to primeChlamydia-specific T cells that could subsequently be cultured from thespleen (Starnbach et al., Infect. Immunol. 63:3527-3530, 1995) and hasalso been demonstrated to cause salpingitis in mice (Tuffrey et al., Br.J. Exp. Pathol. 67:605-16, 1986; Tuffrey et al., J. Exp. Pathol.(Oxford) 71:403-10, 1990). Other studies have used a different Chlamydiaspecies, Chlamydia muridarum, as a mouse model of infection. C.muridarum is not known to infect humans but causes an ascendinginfection when inoculated into the vaginal vault of female mice. Wewere, however, unable to use this model, as our Cta1-specific T cellsdid not recognize C. muridarum-infected cells (data not shown),suggesting the Cta1 epitope in C. trachomatis may not be conserved inthe Cta1 homolog in C. muridarum.

Using intrauterine infection with a human serovar of C. trachomatis,Chlamydia-specific T cells exhibited a Th1 response in the genital tractin response to infection. These cells secreted IFNγ while still in theILNs, and secretion continued following migration into the infectedgenital tract. IFNγ has long been implicated as a critical effector inChlamydia clearance, but may also be a cause of the tissue pathologyassociated with infection. In vitro, IFNγ enhances the ability ofphagocytes to control Chlamydia replication (Rottenberg et al., Curr.Opin. Immunol. 14:444-451, 2002). In vivo, susceptibility to Chlamydiainfection is increased in IFNγ−/− mice and Chlamydia-specific T cellstransferred into mice only appear to be protective if they secrete IFNγ(Loomis et al., Curr. Opin. Microbiol. 5:87-91, 2002). Besides its rolein protection, IFNγ induces Chlamydia to develop into a persistent statein vitro (Rottenberg et al., Curr. Opin. Immunol. 14:444-451, 2002;Hogan et al., Infect. Immunol. 72:1843-1855, 2004.), and there isevidence that organisms persist in some human infections (Villareal etal., Arthritis. Res. 4:5-9, 2002). Persistence or repeated infectionwith Chlamydia may contribute to tissue scarring in vivo (Beatty et al.,Microbiol. Rev. 58:686-699, 1994). Consistent with the hypothesis thatIFNγ may promote tissue pathology, lymphocytes from patients withChlamydia-associated tubal factor infertility secreted high levels ofIFNγ in response to Chlamydia relative to lymphocytes from controlpatients (Kinnunen et al., Clin. Exp. Immunol. 131:299-303, 2003).

In our model, upregulation of CD69 on NR1 T cells in the ILNs did notoccur until three days following genital infection and proliferation didnot occur until four days following the infection. The period betweenintrauterine Chlamydia inoculation and activation of NR1 cells maydefine the amount of time required for Chlamydia antigens to travel intothe draining lymph nodes where the antigen can activate naïve T cells.This timing was significantly later than the proliferation induced inthe spleen after systemic infection (data not shown). Elements of theimmune system in the genital tissues are less well-characterized thanthose in intestinal tissues, but differences between these two mucosalsurfaces are nonetheless apparent. Unlike the intestinal lumen, thegenital mucosa lacks organized lymphoid elements (Parr et al., Biol.Reprod. 44:491-498, 1991; Nandi et al., Reg. Immunol. 5:332-338, 1993).While the intestinal lumen is equipped with Peyer's Patches that canimmediately sample luminal contents, the initiation of T lymphocyteresponses against genital pathogens must occur outside the genitalmucosa, perhaps in the ILNs, which drain antigen from the genital tract(Parr et al., J. Reprod. Immunol. 17:101-14, 1990; Cain et al., Infect.Immunol. 63:1784-9, 1995; Hawkins et al., Infect. Immunol. 68:5587-94,2000; Nandi et al., Reg. Immunol. 5:332-338, 1993). For example, T cellsspecific for the enteric pathogen S. enterica have been shown to beactivated in the Peyer's Patches a few hours after oral infection, and Tcells in these nodes proliferated extensively by two days post-infection(McSorley et al., Immunity 16:365-377, 2002). The amount of time ittakes Chlamydia antigens to migrate from the genital surface to the ILNsmay explain the lack of NR1 activation prior to three dayspost-infection.

The genital and intestinal mucosa also differ in cell surface adhesionmolecules responsible for recruiting lymphocytes. Whereas interaction ofthe α4β7 integrin on lymphocytes with the MAdCAM-1 adhesion molecule onthe intestinal endothelium mediates recruitment of T lymphocytes to theintestinal mucosa, such an interaction does not appear to play asignificant role in recruitment to the genital mucosa (Rott et al., J.Immunol. 156:3727-2736, 1996; Perry et al., J. Immunol. 160:2905-2914,1998).

It has been previously demonstrated that T cells can protect miceagainst Chlamydia infection (Starnbach et al., J. Immunol., 153:5183-9,1994; Starnbach et al., J. Immunol., 171:4742-9, 2003). However,previously it was not been possible to determine when and where naïveChlamydia-specific T cells first encounter antigens, how they trafficfollowing activation, and when and where they proliferate. A majorlimitation in analyzing these early events is the low precursorfrequency of naïve Chlamydia-specific T cells. However, we have nowgenerated the first tool to study the response of Chlamydia-specificnaïve T cells—a T cell receptor (TCR) transgenic mouse.

Chlamydia specific CD4⁺ T cell clone. To identify a Chlamydia-specificTCR for the creation of TCR transgenic mice, we generated aChlamydia-specific CD4⁺ T cell clone named NR9.2. The clone specificallysecreted IFNγ when co-cultured with Chlamydia-infected macrophages in anintracellular cytokine staining assay (FIG. 13A). In addition, adoptivetransfer of this clone protected naïve mice from Chlamydia infection(FIG. 13B). A library of proteins expressed by the C. trachomatis ORFscontained in the genome database was screened. Of the 894 ORFs wescreened, only E. coli expressing the protein encoded by CT788 (FIG. 14)stimulated NR9.2 to secrete significant levels of IFNγ (FIG. 15). CT788was annotated in the published C. trachomatis genome as a predictedperiplasmic protein of unknown function (Stephens et al., Science282:754-759, 1998), and its sequence shares little homology withproteins outside of the Chlamydia genus. CT788 was designated Cta1(Chlamydia-specific T cell antigen-1).

Mice expressing the TCR from the NR9.2 clone. We then generated miceexpressing the TCR from the NR9.2 clone. Peripheral blood mononuclearcells from the resulting TCR transgenic mice expressed the same variablechain elements (Vα2, Vβ8.3) as the T cell clone from which they werederived. We cloned the rearranged genomic TCRα and TCRβ sequences fromNR9.2 into expression vectors and injected these constructs into C57BL/6fertilized oocytes. Pseudopregnant female recipients were then implantedwith the oocytes and individual pups born from the foster mothers werescreened using primers specific for the NR9.2 TCR. A TCR tg founder linewas identified and designated NR1. To confirm that the NR9.2 TCR wasexpressed on the transgenic cells in NR1, cells from the peripheralblood of these animals were tested for expression of the Vα2 and Vβ8.3TCR elements. Vα2 and Vβ8.3 were the variable chains expressed by theoriginal NR9.2 T cell clone (data not shown). A significant percentageof CD4⁺ T cells from the peripheral blood of the transgenic miceexpressed Vα2 and Vβ8.3 (FIG. 16A), demonstrating that both the TCRα andTCRβ transgenes from NR9.2 were efficiently expressed. The transgeniccells were also CD69^(lo), CD25^(lo), CD62L^(lo), CD44^(lo), andCTLA4^(lo), indicating that they were naïve T cells (data not shown).

To determine whether the NR1 transgenic T cells were specific andresponsive to Cta1, we tested the proliferation of transgenic spleencells in response to Cta1₁₃₃₋₁₅₂ (see FIG. 14; SEQ ID NO:2). Spleencells from naïve NR1 mice showed a strong proliferative response toCta1₁₃₃₋₁₅₂ (FIG. 16B). NR1 spleen cells also secreted high levels ofIFNγ in response to this peptide (data not shown). In contrast, spleencells from NR1 did not proliferate in response to a control peptide fromovalbumin, OVA₃₂₃₋₃₃₆ (data not shown).

To determine whether the NR1 transgenic T cells responded to Chlamydiain vivo, thirty million cells from the spleen and peripheral lymph nodesof NR1 mice were labeled with the fluorescent dye carboxyfluoresceindiacetate succinimidyl ester (CFSE) and adoptively transferred intoC57BL/6 recipients. As approximately 10% of NR1 cells were CD4⁺ T cellsexpressing the Cta1-specific TCR (data not shown), the transferredpopulation contained approximately 3×10⁶ Cta1-specific T cells.CFSE-labeled NR1 transgenic cells retained high levels of CFSE followingtransfer into uninfected recipient mice, indicating that the transgeniccells did not divide in the absence of infection.

In other C57BL/6 animals that had received the CFSE-labeled transgeniccells, the animals were infected intravenously with 10⁷ IFU of C.trachomatis. The C. trachomatis organisms used for infection wereserovar L2, which is associated with lymphogranuloma venereum (LGV) inhumans, or serovar D, which is associated with typical human genitaltract infection. Within three days of infection with either C.trachomatis serovar, the transgenic cells had proliferated extensively(FIG. 17). We also observed that the proliferation of NR1 cells wasspecific for C. trachomatis infection. When animals that had receivedNR1 cells were infected intravenously with Salmonella enterica orListeria monocytogenes, the transgenic T cells were not stimulated toproliferate. As an additional control to demonstrate that TCR tg T cellswith other specificities would not respond to C. trachomatis infectionin the recipient mice, we showed that ovalbumin-specific OTH transgenicT cells proliferated in response to ovalbumin protein with adjuvant(data not shown) but not in response to C. trachomatis infection (FIG.17).

To study CD4⁺ T cell responses to C. trachomatis in the context of amucosal infection, Thy1.1 (CD90.1) recipient mice adoptively transferredwith CFSE-labeled transgenic cells were infected in the uterine hornswith C. trachomatis L2. Using Thy1.1 recipient mice, donor transgeniccells were readily distinguished from endogenous cells in the recipientanimals. The activation status of the transgenic cells was assessed byexamining cell surface expression of the early activation marker CD69and the naïve T cell marker CD62L on T cells from iliac lymph nodes(ILNs), which drain antigen from the genital tract (Parr et al., J.Reprod. Immunol. 17:101-14, 1990; Cain et al., Infect. Immunol.63:1784-9, 1995; Hawkins et al., Infect. Immunol. 68:5587-94, 2000). TheILNs were removed five days after infection and NR1 T cells in the nodeswere examined for CD69 upregulation and loss of CFSE fluorescence.Transgenic cells in uninfected mice were predominantly not activated(CD69^(lo)) and of the naïve phenotype (CD62L^(hi)). Followinginfection, CD69 was upregulated on cells that had undergone a few roundsof cell division and downregulated on cells that had undergone furtherrounds of division. CD62L was downregulated on a sub-population oftransgenic cells that had extensively divided (FIG. 18A). Whereas T cellproliferation was weak in the non-draining lymph nodes, extensive T cellproliferation was observed four days post-infection in the draininglymph nodes (FIG. 18B). After activation and proliferation, transgeniccells were also recruited to the genital mucosa in infected mice (FIG.18C).

As shown in FIG. 18A, a significant number of NR1 T cells from infectedanimals showed progressive dilution of CFSE, suggesting that extensiveproliferation had occurred. Furthermore, recently divided (CFSE^(med))NR1 T cells expressed high levels of CD69, indicating that these cellsalso had been recently activated. Once NR1 cells had undergone extensiveproliferation (CFSE^(lo)), they expressed lower levels of CD69. Theseresults are consistent with CD69 as an early T cell activation markerthat is only transiently upregulated following antigen encounter(Ziegler et al., Stem Cells 12:456-65, 1994; Cochran et al., Immunity12:241-50, 2000). Cells from the ILNs of mock infected recipients wereCFSE^(hi) and had not upregulated CD69, suggesting that they were notactivated. Interestingly, there was a similar population ofCFSE^(hi)CD69^(lo) NR1 cells in the infected recipients. These could becells that did not encounter antigen, or they could be cells that werenot expressing the appropriate Cta1-specific TCR because of endogenousTCR rearrangements (von Boehmer, Annu. Rev. Immunol. 8:531-556,1990;Balomenos et al., J. Immunol. 155:3308-3312, 1995).

Other characteristics of activated T cells include downregulation of thenaive marker CD62L and upregulation of the activation molecule CD44. Toconfirm that NR1 cells were activated following Chlamydia genitalinfection, the expression of CD62L and CD44 on transferred CFSE-labeledNR1 T cells from the ILNs of recipient mice was analyzed. Seven daysafter infection, a subset of NR1 T cells that had proliferatedextensively (CFSE^(lo)) had reduced expression of CD62L (FIG. 18A). Bycontrast, undivided (CFSE^(hi)) NR1 cells in both mock infected andinfected recipients were mostly CD62L^(hi). In addition to displaying aCD62L^(lo) phenotype, effector T cells typically express high levels ofthe activation marker CD44. Proliferating NR1 T cells from the ILNs ofinfected recipients were exclusively CD44^(hi) (FIG. 18A). Thus, NR1cells were activated and proliferated in the ILNs of mice followinggenital infection with Chlamydia.

Extensive proliferation of NR1 cells occurs preferentially in the ILNs.To confirm that activation and proliferation of NR1 cells in the ILNsresulted from antigen draining from the genital tract to these nodes,the response of NR1 cells in the ILNs was compared to the response inthe lymph nodes that do not drain the genital tract (nondraining lymphnodes, NDLNs). T cell activation and proliferation was monitored overtime to ensure that any activity that may have occurred in the NDLNsover the course of infection would be observed. 3×10⁷ CFSE-labeled NR1cells were transferred into CD90.1 mice. The mice were then infected inthe uterus with 10⁶ IFU C. trachomatis serovar L2. Lymph nodes were thenharvested at various times following infection. In the ILNs,upregulation of CD69 was seen on NR1 cells beginning three days afterinfection. Four days after infection, downregulation of CD62L andupregulation of CD44 was observed. Acquisition of the activation markersoccurred preferentially in NR1 cells from the ILNs and not in NR1 cellsfrom the NDLNs (data not shown). Thus, NR1 cells were specificallyactivated in the ILNs following genital infection with Chlamydia.

By monitoring proliferation of the transferred NR1 cells at varioustimes following infection, we confirmed that NR1 cells preferentiallyencountered antigen in the ILNs. In the ILNs, NR1 cells werepredominantly CFSE^(hi) two and three days post infection, suggestingthat these cells had not proliferated at these early time points (FIG.19), but NR1 cells in the ILNs had proliferated extensively within fourdays of infection. While NR1 cells had also proliferated in the NDLNswithin four days of infection, the number was significantly less thanthat seen in the ILNs. The proliferating NR1 cells in the NDLNscontained lower levels of CFSE and did not express significant levels ofCD69 relative those in the ILNs (data not shown), suggesting that thesecells migrated to the NDLNs from other sites following activation.Significant expansion of NR1 T cells in the NDLNs did not occur even oneweek after infection (data not shown), again demonstrating thatproliferation of NR1 cells occurred preferentially in the ILNs.

NR1 cells in the ILNs develop the ability to secrete IFNγ. To examinethe differentiation of antigen-activated NR1 cells into effector Tcells, we determined that proliferating NR1 cells secreted effectorcytokines in response to C. trachomatis infection. 3×10⁷ CFSE-labeledNR1 cells were transferred into CD90.1 congenic mice, and then thesemice were infected in the uterus with 10⁶ IFU of C. trachomatis serovarL2. The ILNs were removed six days later, and the NR1 T cells wereanalyzed by flow cytometry for production of cytokines. ProliferatingNR1 cells) (CFSE^(lo)) secreted IFNγ whereas non-proliferating cells(CFSE^(hi)) did not secrete IFNγ (FIG. 20). The NR1 cells did notsecrete IL-4 (data not shown). Thus, NR1 cells differentiatedpreferentially into effector T cells of the Th1 phenotype followinggenital infection with Chlamydia.

Antigen-experienced NR1 cells traffic to the genital tract. Followingactivation, effector T cells are typically recruited to the site ofinfection where they contribute to the elimination of the pathogen(Swain et al., Adv. Exp. Med. Biol. 512:113-120, 2002; Gallichan et al.,J. Exp. Med. 184:1879-1890, 1996). To determine whetherChlamydia-specific NR1 cells migrated to the site of genital infectionin mice, 3×10⁷ CFSE-labeled NR1 cells were transferred into CD90.1congenic mice. These recipients were infected in the uterus with 10⁶ IFUof C. trachomatis serovar L2. Genital tract tissue was then isolated andthe presence of transferred Chlamydia-specific T cells was determined.Significantly more NR1 cells were observed in the genital mucosa ofanimals infected with C. trachomatis than in animals that were mockinfected (FIG. 18C). Transgenic cells that were recruited to the genitaltract had the phenotype of antigen-experienced cells (CFSE^(lo)CD62L^(lo) CD44^(hi)) and secreted IFNγ (FIGS. 21A and 21B). In summary,activated NR1 TCR tg T cells that secrete IFNγ are recruited to thegenital mucosa in response to C. trachomatis infection.

Antigenic Fragments of Cta1

To map more closely the epitope within Cta1 recognized by the NR9.2 Tcells, a series of overlapping 20-mer peptides covering the Cta1sequence for their ability to stimulate NR9.2 to secrete IFNγ werescreened (Table 2). The 20-mer peptide Cta1₁₃₃₋₁₅₂(KGIDPQELWVWKKGMPNWEK; SEQ ID NO:2) induced NR9.2 to secrete significantlevels of IFNγ (FIG. 22), confirming the specificity of these C.trachomatis-specific T cells to an epitope within these 20 amino acids.

TABLE 2  Antigen/T cell line, peptide, and IFNγ measured (ng) (SEQ ID NOS: 2 and 156-171) Cta1/NR9.2 LESTSLYKKAGCANKKNRNL0.4 (±0.2) GCANKKNRNLIGWFLAGMFF 0.5 (±0.3) IGWFLAGMFFGIFAIIFLLI0.6 (±0.3) GIFAIIFLLILPPLPSSTQD 0.4 (±0.2) LPPLPSSTQDNRSMDQQDSE0.6 (±0.2) NRSMDQQDSEEFLLQNTLED 0 6 (+0.3) EFLLQNTLEDSEIISIPDTM0.6 (±0.2) SEIISIPDTMNQIAIDTEKW 0.6 (±0.2) NQIAIDTEKWFYLNKDYTNV0.5 (±0.2) FYLNKDYTNVGPISIVQLTA 0.7 (±0.4) GPISIVQLTAFLKECKHSPE0.5 (±0.3) FLKECKHSPEKGIDPQELWV 0.5 (±0.4) KGIDPQELWVWKKGMPNWEK58.0 (±14.0) WKKGMPNWEKVKNIPELSGT 1.4 (±0.2) VKNIPELSGTVKDESPSFLV0.8 (±0.1) VKDESPSFLVQSGVAGLEQL 0.7 (±0.2) QSGVAGLEQLESI 0.8 (±0.2)By screening deletion peptides of this 20-mer, a minimal sequencerequired for activation of the NR9.2 can be identified. Epitopicsequences can include 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, and 19 amino acid fragments of Cta1₁₃₃₋₁₅₂ and can includeN-terminal or C-terminal deletions of the Cta1₁₃₃₋₁₅₂ fragment, or acombination of N- and C-terminal deletions (see, e.g., Table 1).Further, antigenic peptides in other regions of Cta1 or using other Tcell clones can also be identified similarly. Compositions and methodsof the invention may include or employ combinations of epitopicfragments described herein. Antigenicity of Cta1 fragments can bedetermined using methods known in the art and methods described herein.

The experiments described above were performed as follows.

Mice. C57BL/6J (H-2^(b)), B6.PL-thy1^(a)0Cy (CD90.1 congenic), and OTHmice were obtained from the Jackson Laboratory.

Tissue culture. Bone marrow derived dendritic cells (BMDCs) and bonemarrow derived macrophages (BMMs) were cultured as previously described(Steele et al., J. Immunol. 173:6327-6337, 2004; Shaw et al., Infect.Immunol. 74:1001-1008, 2006).

Growth, isolation, and detection of bacteria. Elementary bodies (EBs) ofC. trachomatis serovar L2 434/Bu and C. trachomatis serovar D (UW-3/Cs)were propagated and quantitated as previously described (Starnbach etal., J. Immunol. 171:4742-4749, 2003). Salmonella enterica serovarTyphimurium (ATCC 14028) was grown at 37° C. in Luria-Bertani (LB)medium. Listeria monocytogenes 10403s was grown at 30° C. in brain heartinfusion (BHI) medium (Difco/Becton Dickinson, Sparks, Md.).

Generation of the NR9.2 T cell clone. Splenocytes from mice wereisolated 21 days after infection with C. trachomatis serovar L2 and werecultured with irradiated (2,000 rads) BMDCs, UV-inactivated C.trachomatis serovar L2, and naïve syngeneic splenocytes in RP-10 (RPMI1640 supplemented with 10% FCS, L-glutamine, HEPES, 50 μM 2-βME, 50 U/mlpenicillin, and 50 μg/ml streptomycin) with α-methyl mannoside and 5%supernatant from concanavalin A-stimulated rat spleen cells. CD8⁺ Tcells were depleted from the culture using Dynabeads Mouse CD8(Invitrogen). The CD4⁺ T cells were restimulated every seven days withC. trachomatis-pulsed BMDCs. Once a C. trachomatis-specific CD4⁺ T cellline was established, the T cell clone NR9.2 was isolated by limitingdilution.

Identification of the T cell antigen Cta1. An expression library ofgenomic sequences from C. trachomatis serovar D was inserted into amodified form of the pDEST17 vector (Invitrogen) and transformed intothe Stbl2 strain of E. coli (Invitrogen). Following induction of proteinexpression, the bacteria were fixed in 0.5% paraformaldehyde andincubated with BMMs. NR9.2 T cells were then added and after 24 h, thesupernatant was tested for the level of IFNγ by ELISA (Endogen,Rockford, Ill.). To identify the reactive peptide epitope within Cta1,synthetic 20-mer peptides (MIT Biopolymers Lab, Cambridge, Mass.) wereused at a concentration of 25 μM in an IFNγ ELISA (Endogen).

Flow cytometry and antibodies. Antibodies specific for CD4, CD90.2, Vα2,Vβ8.3, CD69, CD25, CD44, CD62L, CTLA-4, IFNγ, and IL-4 were purchasedfrom BD Biosciences (San Diego, Calif.). Data were collected on a BDBiosciences FACSCalibur™ flow cytometer (San Jose, Calif.) and analyzedusing CellQuest™ software. Intracellular cytokine staining was performedby incubating NR9.2 T cells with Chlamydia-infected BMMs (MOI 5:1) inthe presence of GolgiPlug™ reagent (BD Biosciences). Intracellularcytokine staining of NR1 transgenic cells was performed by stimulatingcells for 4 hours in the presence of PMA (50 ng/ml, MP Biomedicals,Solon, Ohio), ionomycin (1 μg/ml, Sigma, St. Louis, Mo.), and GolgiPlug™reagent (BD Biosciences). Cells were permeabilized with theCytofix/Cytoperm Plus kit (BD Biosciences). PE-conjugated rat IgG1 (BDBiosciences) was used as an isotype control antibody.

Generation of NR1 TCR tg mice. The rearranged TCR from NR9.2 uses theVα2Jα16 and Vβ8.3DJβ1.2 receptor chains. The genomic TCR sequences werecloned and inserted into the TCR vectors pTα and pTβ at the recommendedrestriction sites (Kouskoff et al., J. Immunol. Methods 180:273-80,1995). Prokaryotic DNA sequences were then removed from both vectorsprior to injection into the pronuclei of fertilized C57BL/6J oocytes.TCR transgenic founders were identified by PCR. Routine screening toidentify transgenic mice was carried out by staining samples of orbitalblood from the mice with antibodies specific for Vα2 and Vβ8.3, followedby flow cytometry.

Adoptive transfer of NR1 cells, infection of mice, and preparation oftissues from mice. Spleen and peripheral lymph nodes were isolated fromNR1 TCR tg mice and labeled with the dye CFSE (5 μM, Molecular Probes,Eugene, Oreg.). Recipient mice were injected i.v. with 3×10⁷ NR1 cells.Mice were infected one day after transfer of the cells. Where indicated,mice were infected intravenously with 10⁷ IFU of C. trachomatis, 3×10³CFU of L. monocytogenes, or 5×10³ CFU of S. enterica. To infect thegenital tract, mice were treated with 2.5 mg of medroxyprogesteroneacetate subcutaneously one week prior to infection to synchronize themice into a diestrus state (Perry et al., Infect. Immunol. 67:3686-3689,1999; Ramsey et al., Infect. Immunol. 67:3019-3025, 1999). Intrauterineinfection was carried out by inoculating the uterine horns with 10⁶ IFUof C. trachomatis serovar L2. At various times after infection,single-cell suspensions of spleen, ILNs, or NDLNs taken from theaxillary and cervical lymph nodes were prepared, stained, and analyzedby flow cytometry as described above. To isolate lymphocytes from thegenital mucosa, genital tracts (oviduct, uterus, and cervix) wereremoved from mice and digested with collagenase (type XI, Sigma) for onehour prior to staining and flow cytometry.

Intracellular cytokine staining. Bone marrow-derived macrophages wereinfected with C. trachomatis L2 at an MOI of 5:1 for 16-18 hours. Tcells were added at an effector-to-target ratio of 5:1 and incubated foranother 6 hours in the presence of brefeldin A (Pharmingen). Cells werepermeabilized and stained for the presence of IFNγ. Cells were thenanalyzed using standard flow cytometric techniques on a FACScan flowcytometer.

Protection assay. Ten days after T cell restimulation, 10⁷ T cells werewashed twice with PBS and adoptively transferred into C57BL/6 recipientmice. Mice were then infected i.v. with 10⁷ IFUs C. trachomatis L2.Spleens were titered three days later on a McCoy cell monolayer. Ascontrols, naïve mice and Chlamydia-immune mice were also infected withC. trachomatis L2 and spleens were titered three days later.

Intrauterine infection. Intrauterine infection with C. trachomatis wascarried out by surgical inoculation of the uterine horns with 10⁶ IFUsC. trachomatis L2. Draining (iliac) and non-draining (axillary,cervical, mandibular) lymph nodes were harvested and single cellsuspensions were analyzed using standard flow cytometric techniques on aFACScan flow cytometer.

CT788 Polypeptide Expression

A CT788 polypeptide or fragment thereof may be produced bytransformation of a suitable host cell with all or part of apolypeptide-encoding polynucleotide molecule or fragment thereof in asuitable expression vehicle.

Those skilled in the field of molecular biology will understand that anyof a wide variety of expression systems may be used to provide thepolypeptide CT788 or fragment thereof. The precise host cell used is notcritical to the invention. The CT788 polypeptide or fragment thereof maybe produced in a prokaryotic host (e.g., E. coli) or in a eukaryotichost (e.g., Saccharomyces cerevisiae, insect cells, e.g., Sf21 cells, ormammalian cells, e.g., NIH 3T3, HeLa, or preferably COS cells). Suchcells are available from a wide range of sources (e.g., the AmericanType Culture Collection, Rockland, Md.; also, see, e.g., Ausubel et al.,supra). The method of transformation or transfection and the choice ofexpression vehicle will depend on the host system selected.Transformation and transfection methods are described, e.g., in Ausubelet al. (supra); expression vehicles may be chosen from those provided,e.g., in Cloning Vectors: A Laboratory Manual (Pouwels, P. H. et al.,1985, Supp. 1987).

Once the recombinant CT788 polypeptide or fragment thereof is expressed,it is isolated, e.g., using affinity chromatography. In one example, anantibody raised against a CT788 polypeptide or fragment thereof may beattached to a column and used to isolate the recombinant polypeptide.Lysis and fractionation of polypeptide-harboring cells prior to affinitychromatography may be performed by standard methods (see, e.g., Ausubelet al., supra).

Once isolated, the recombinant CT788 polypeptide or fragment thereofcan, if desired, be further purified, e.g., by high performance liquidchromatography (see, e.g., Fisher, Laboratory Techniques In BiochemistryAnd Molecular Biology, eds., Work and Burdon, Elsevier, 1980).

Short fragments of CT788, can also be produced by chemical synthesis(e.g., by the methods described in Solid Phase Peptide Synthesis, 2nded., 1984 The Pierce Chemical Co., Rockford, Ill.).

Also included in the invention are CT788 proteins or fragments thereoffused to heterologous sequences, such as detectable markers (forexample, proteins that may be detected directly or indirectly such asgreen fluorescent protein, hemagglutinin, or alkaline phosphatase), DNAbinding domains (for example, GAL4 or LexA), gene activation domains(for example, GAL4 or VP16), purification tags, or secretion signalpeptides. These fusion proteins may be produced by any standard method.Fusion protein may also include fusions to immunogenic proteins such asan Ig protein, e.g., as IgG, IgM, IgA, or IgE or the Fc region of an Igprotein. For production of stable cell lines expressing a CT788 fusionprotein, PCR-amplified CT788 nucleic acids may be cloned into therestriction site of a derivative of a mammalian expression vector. Forexample, KA, which is a derivative of pcDNA3 (Invitrogen, Carlsbad,Calif.) contains a DNA fragment encoding an influenza virushemagglutinin (HA). Alternatively, vector derivatives encoding othertags, such as c-myc or poly Histidine tags, can be used.

Other sequences that may be fused to CT788 include those that provideimmunostimulatory function, such as interleukin-2 (Fan et al., ActaBiochim. Biophys. Sin. 38:683-690, 2006), Toll-like receptor-5 flagellin(Huleatt et al., Vaccine 8:763-775, 2007), simian immunodeficiency virusTat (Chen et al., Vaccine 24:708-715, 2006), or fibrinogen-albumin-IgGreceptor of group C streptococci (Schulze et al., Vaccine 23:1408-1413,2005). In addition, heterologous sequences may be added to enhancesolubility or increase half-life, for example, hydrophilic amino acidresidues (Murby et al., Eur. J. Biochem. 230:38-44, 1995), glycosylationsequences (Sinclair and Elliott, J. Pharm. Sci. 94:1626-1635, 2005), orthe carboxy terminus of human chorionic gonadotropin or thrombopoeitin(Lee et al., Biochem. Biophys. Res. Comm. 339:380-385, 2006).

Vaccine Production

The invention also provides for a vaccine composition including a CT788polypeptide, an fragment (e.g., an immunogenic fragment) of CT788, anypolypeptide identified in a method of the invention, or an fragment(e.g., an immunogenic fragment) thereof. The vaccine may further includean additional antigenic peptide fragment (e.g., 2, 3, 4, 5, 6, 10, ormore different fragments). The invention further includes a method ofinducing an immunological response in an individual, particularly ahuman, the method including inoculating the individual with a CT788polypeptide or a fragment or fragments thereof (e.g., an immunogenicfragment), in a suitable carrier for the purpose of inducing an immuneresponse to protect an individual from infection, particularly bacterialinfection, and most particularly Chlamydia infection. The administrationof this immunological composition may be used either therapeutically inindividuals already experiencing an infection, or may be usedprophylactically to prevent an infection.

The preparation of vaccines that contain immunogenic polypeptides isknown to one skilled in the art. The CT788 polypeptide, fragment of aCT788 polypeptide, polypeptide identified in a method of the invention,or fragment thereof may serve as an antigen for vaccination, or anexpression vector encoding the polypeptide, or fragments or variantsthereof, might be delivered in vivo in order to induce an immunologicalresponse comprising the production of antibodies or, in particular, a Tcell immune response.

A CT788 polypeptide, polypeptide identified in a method of theinvention, or fragment or variant thereof may be fused to a recombinantprotein that stabilizes the polypeptide, aids in its solubilization,facilitates its production or purification, or acts as an adjuvant byproviding additional stimulation of the immune system. The compositionsand methods comprising the polypeptides or nucleotides of the inventionand immunostimulatory DNA sequences are described in (Sato et al.,Science 273:352, 1996).

Typically vaccines are prepared in an injectable form, either as aliquid solution or as a suspension. Solid forms suitable for injectionmay also be prepared as emulsions, or with the polypeptides encapsulatedin liposomes. Vaccine antigens are usually combined with apharmaceutically acceptable carrier, which includes any carrier thatdoes not induce the production of antibodies harmful to the individualreceiving the carrier. Suitable carriers typically comprise largemacromolecules that are slowly metabolized, such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, lipid aggregates, and inactive virusparticles. Such carriers are well known to those skilled in the art.These carriers may also function as adjuvants.

Adjuvants are immunostimulating agents that enhance vaccineeffectiveness. Effective adjuvants include, but are not limited to,aluminum salts such as aluminum hydroxide and aluminum phosphate,muramyl peptides (e.g., muramyl dipeptide), bacterial cell wallcomponents, saponin adjuvants, and other substances that act asimmunostimulating agents to enhance the effectiveness of thecomposition. Other adjuvants include liposomal formulations, syntheticadjuvants, such as saponins (e.g., QS21), monophosphoryl lipid A, andpolyphosphazine.

Immunogenic compositions of the invention, e.g., the CT788 polypeptide,a CT788 fragment, polypeptide identified in a method of the invention,or a fragment thereof, a pharmaceutically acceptable carrier, andadjuvant, also typically contain diluents, such as water, saline,glycerol, ethanol. Auxiliary substances may also be present, such aswetting or emulsifying agents, pH buffering substances, and the like.Proteins may be formulated into the vaccine as neutral or salt forms.The vaccines are typically administered parenterally, by injection; suchinjection may be subcutaneous, intradermal, intramuscular, intravenous,or intraperitoneal. Additional formulations are suitable for other formsof administration, such as by suppository or orally. Oral compositionsmay be administered as a solution, suspension, tablet, pill, capsule, orsustained release formulation. Vaccines may also be administered by anocular, intranasal, gastric, pulmonary, intestinal, rectal, vaginal, orurinary tract route. The administration can be achieved in a single doseor repeated at intervals. The appropriate dosage depends on variousparameters that are understood by those skilled in the art, such as thenature of the vaccine itself, the route of administration, and thecondition of the mammal to be vaccinated (e.g., the weight, age, andgeneral health of the mammal).

In addition, the vaccine can also be administered to individuals togenerate polyclonal antibodies (purified or isolated from serum usingstandard methods) that may be used to passively immunize an individual.These polyclonal antibodies can also serve as immunochemical reagents.

Antigenic peptides (e.g., CT788 peptides such as those described hereinincluding CT788₁₃₃₋₁₅₂ or any fragment thereof) may also be administeredas fusion peptides. For example, a polypeptide or polypeptide derivativemay be fused to a polypeptide having adjuvant activity, such as, e.g.,subunit B of either cholera toxin or E. coli heat-labile toxin. Severalpossibilities are can be used for achieving fusion. First, thepolypeptide of the invention can be fused to the N- or C-terminal end ofthe polypeptide having adjuvant activity. Second, a polypeptide fragmentcan be fused within the amino acid sequence of the polypeptide havingadjuvant activity.

Pharmaceutical Compositions

In addition to vaccines, the invention also provides pharmaceuticalcompositions that include a polypeptide or a fragment thereof identifiedusing a method of the invention or compositions that include at CT788polypeptide or a fragment thereof (e.g., an immunogenic fragment or anyfragment described herein). Such compositions may be incorporated into apharmaceutical composition, dispersed in a pharmaceutically-acceptablecarrier, vehicle or diluent. In one embodiment, the pharmaceuticalcomposition includes a pharmaceutically-acceptable excipient. Thecompounds of the present invention may be administered by any suitablemeans, depending, for example, on their intended use, as is well knownin the art, based on the present description. For example, if compoundsof the present invention are to be administered orally, they may beformulated as tablets, capsules, granules, powders or syrups.Alternatively, formulations of the present invention may be administeredparenterally as injections (intravenous, intramuscular or subcutaneous),drop infusion preparations or suppositories. For application by theophthalmic mucous membrane route, compounds of the present invention maybe formulated as eye drops or eye ointments. These formulations may beprepared by conventional means, and, if desired, the compounds may bemixed with any conventional additive, such as an excipient, a binder, adisintegrating agent, a lubricant, a corrigent, a solubilizing agent, asuspension aid, an emulsifying agent or a coating agent.

Subject compounds may be suitable for oral, nasal, topical (includingbuccal and sublingual), rectal, vaginal, aerosol and/or parenteraladministration. The formulations may conveniently be presented in unitdosage form and may be prepared by any methods well known in the art ofpharmacy. The amount of agent that may be combined with a carriermaterial to produce a single dose vary depending upon the subject beingtreated, and the particular mode of administration.

Pharmaceutical compositions of this invention suitable for parenteraladministration includes one or more components of a supplement incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or non-aqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Methods of Treating a Pathogenic Disease or Neoplasm

The polypeptides, vaccines, and pharmaceutical compositions describedherein may be used in a variety of treatments of diseases including apathogenic infection and in the treatment of a neoplastic disorder.Those skilled in the art will understand, the dosage of any compositiondescribed herein will vary depending on the symptoms, age and bodyweight of the patient, the nature and severity of the disorder to betreated or prevented, the route of administration, and the form of thesupplement. Any of the subject formulations may be administered in anysuitable dose, such as, for example, in a single dose or in divideddoses. Dosages for the compounds of the present invention, alone ortogether with any other compound of the present invention, or incombination with any compound deemed useful for the particular disorder,disease or condition sought to be treated, may be readily determined bytechniques known to those of skill in the art. Also, the presentinvention provides mixtures of more than one subject compound, as wellas other therapeutic agents.

The combined use of several compounds of the present invention, oralternatively other therapeutic agents, may reduce the required dosagefor any individual component because the onset and duration of effect ofthe different components may be complimentary. In such combined therapy,the different active agents may be delivered together or separately, andsimultaneously or at different times within the day.

Therapeutic Antibodies and T-Cell Depletion

Alternatively, the immune response to Chlamydia, rather than theinfection itself, may be responsible for symptoms which accompanyinfection, including sterility and pelvic inflammatory disease. In thiscase, it may be desirable to limit the immune response by a subset ofCD4⁺ or CD8⁺ T-cells within an infected individual. Antibodies targetedtowards T-cell clones identified in the methods of the invention (e.g.,antibodies specific to CD4⁺ cells targeted to CT788) may therefore beuseful in treating or preventing deleterious effects associated withChlamydia infection. Methods for selective depletion of specificpopulations of T-cell are described, for example, in Weinberg et al.,Nat Med. 2(2):183-189, 1996.

All patents, patent applications, and publications mentioned in thisspecification are herein incorporated by reference to the same extent asif each independent patent, patent application, or publication wasspecifically and individually indicated to be incorporated by reference.

What is claimed is: 1-14. (canceled)
 15. A vaccine comprising at leastone epitope identified by a method of determining whether a librarycontains or encodes an immunogenic polypeptide, wherein said librarycomprises at least 20 discrete members in defined locations and saidmembers each comprise a cell or virus comprising a pre-determined openreading frame of a polynucleotide encoding a polypeptide, or a portionthereof, differentially expressed in a neoplastic cell as compared to acorresponding normal cell, said pre-predetermined open reading frameoperably linked to a promoter, said method comprising: (a) individuallycontacting a member of said library with a second cell capable of (i)endocytosing said cell or said virus and (ii) displaying a polypeptideencoded by said pre-determined open reading frame on its surface throughthe major histocompatibility complex (MHC class) I pathway; (b)individually contacting each member of step (a) with a cytotoxic Tlymphocyte (CTL) cell derived from a mammal having, or having previouslyhad, a neoplasm; (c) detecting whether said CTL cell is activated,wherein activation of said CTL cell indicates that said member of saidlibrary contains said pre-determined open reading frame which encodes atleast a portion of said immunogenic polypeptide; and (d) identifying anepitope sufficient for CTL activation within said polypeptide, orportion thereof, determined to be immunogenic in step (c), furthercomprising a pharmaceutically acceptable carrier.
 16. An epitopecocktail comprising at least 5 epitopes identified using a method ofdetermining whether a library contains or encodes an immunogenicpolypeptide, wherein said library comprises at least 20 discrete membersin defined locations and said members each comprise a cell or viruscomprising a pre-determined open reading frame of a polynucleotideencoding a polypeptide, or a portion thereof, differentially expressedin a neoplastic cell as compared to a corresponding normal cell, saidpre-predetermined open reading frame operably linked to a promoter, saidmethod comprising: (a) individually contacting a member of said librarywith a second cell capable of (i) endocytosing said cell or said virusand (ii) displaying a polypeptide encoded by said pre-determined openreading frame on its surface through the major histocompatibilitycomplex (MHC class) I pathway; (b) individually contacting each memberof step (a) with a cytotoxic T lymphocyte (CTL) cell derived from amammal having, or having previously had, a neoplasm; (c) detectingwhether said CTL cell is activated, wherein activation of said CTL cellindicates that said member of said library contains said pre-determinedopen reading frame which encodes at least a portion of said immunogenicpolypeptide; and (d) identifying an epitope sufficient for CTLactivation within said polypeptide, or portion thereof, determined to beimmunogenic in step (c).
 17. A composition comprising at least oneimmunogenic polypeptide, or a portion thereof, identified by a method ofdetermining whether a library contains or encodes an immunogenicpolypeptide, wherein said library comprises at least 20 discrete membersin defined locations and said members each comprise a cell or viruscomprising a pre-determined open reading frame of a polynucleotideencoding a polypeptide, or a portion thereof, differentially expressedin a neoplastic cell as compared to a corresponding normal cell, saidpre-predetermined open reading frame operably linked to a promoter, saidmethod comprising: (a) individually contacting a member of said librarywith a second cell capable of (i) endocytosing said cell or said virusand (ii) displaying a polypeptide encoded by said pre-determined openreading frame on its surface through the major histocompatibilitycomplex (MHC class) I pathway; (b) individually contacting each memberof step (a) with a cytotoxic T lymphocyte (CTL) cell derived from amammal having, or having previously had, a neoplasm; and (c) detectingwhether said CTL cell is activated, wherein activation of said CTL cellindicates that said member of said library contains said pre-determinedopen reading frame which encodes at least a portion of said immunogenicpolypeptide.
 18. A vaccine comprising at least one immunogenicpolypeptide, or a portion thereof, identified by a method of determiningwhether a library contains or encodes an immunogenic polypeptide,wherein said library comprises at least 20 discrete members in definedlocations and said members each comprise a cell or virus comprising apre-determined open reading frame of a polynucleotide encoding apolypeptide, or a portion thereof, differentially expressed in aneoplastic cell as compared to a corresponding normal cell, saidpre-predetermined open reading frame operably linked to a promoter, saidmethod comprising: (a) individually contacting a member of said librarywith a second cell capable of (i) endocytosing said cell or said virusand (ii) displaying a polypeptide encoded by said pre-determined openreading frame on its surface through the major histocompatibilitycomplex (MHC class) I pathway; (b) individually contacting each memberof step (a) with a cytotoxic T lymphocyte (CTL) cell derived from amammal having, or having previously had, a neoplasm; and (c) detectingwhether said CTL cell is activated, wherein activation of said CTL cellindicates that said member of said library contains said pre-determinedopen reading frame which encodes at least a portion of said immunogenicpolypeptide, further comprising a pharmaceutically acceptable carrier.19. The composition of claim 17, wherein said contacting step (b) ofsaid method is performed using a plurality of CTL cells.
 20. Thecomposition of claim 17, wherein said method further comprisescomprising performing said method steps (b) and (c) at least one furthertime using said library.
 21. The composition of claim 20, wherein adifferent CTL cell is contacted in step (b) of said method each timesaid method is performed. 22-38. (canceled)
 48. The composition of claim17, wherein said neoplastic cell is a cancer cell.
 49. The compositionof claim 48, wherein said cancer cell is a breast cancer cell. 50-58.(canceled)
 59. The composition of claim 17, wherein said portion of saidpolypeptide has at least 95% sequence identity to the correspondingportion of the polypeptide differentially expressed in said neoplasticcell.
 60. (canceled)
 61. The composition of claim 17, wherein eachmember of said library comprises a single polynucleotide differentiallyexpressed in said neoplastic cell. 62-73. (canceled)
 74. Apharmaceutical composition comprising polypeptide comprising a CT788polypeptide or comprising an immunogenic fragment of said CT788polypeptide, and a pharmaceutically acceptable carrier. 75-80.(canceled)
 81. The composition of claim 17, wherein said member of saidlibrary further comprises a polynucleotide encoding a pore-formingprotein.
 82. The composition of claim 81, wherein said pore-formingprotein is listeriolysin O.
 83. The composition of claim 17, whereinsaid second cell is a macrophage.
 84. The composition of claim 17,wherein said member of said library is killed prior to said contactingstep (a) of said method.
 85. The composition of claim 17, wherein priorto said contacting step (b) of said method, said second cell is killed.86. The composition of claim 17, wherein, prior to said contacting step(a) of said method, a replica of said library is made.
 87. Thecomposition of claim 17, wherein the method further comprises step: (d)recovering said polypeptide identified in step (c) from a replica copyof said library.